JP2007509042A - Methods for altering hematopoietic progenitor cell adhesion, differentiation and migration - Google Patents

Abstract

The present invention provides a method for altering the adhesion and / or migration of hematopoietic progenitor cells to a target tissue and for altering the differentiation of hematopoietic progenitor cells into secondary cell types. Meeting the needs of The present invention also screens test compounds for altering the level of adhesion and / or migration of hematopoietic progenitor cells to the target tissue and for altering differentiation of hematopoietic progenitor cells into secondary cell types. Providing a method for The invention further provides a method for isolating hematopoietic progenitor cells.

Description

FIELD OF THE INVENTION The present invention is for altering the adhesion and / or migration of hematopoietic progenitor cells (such as hematopoietic stem cells and endothelial progenitor cells) to target tissues, and altering the differentiation of hematopoietic progenitor cells into secondary cells. It relates to a method for making it happen. The invention also provides for screening test compounds for altering the level of adhesion and / or migration of hematopoietic progenitor cells to a target tissue and for altering differentiation of hematopoietic progenitor cells into secondary cells. Regarding the method. The invention further relates to a method for isolating hematopoietic progenitor cells.

  This application claims priority to co-pending US Provisional Patent Application No. 60 / 507,202, filed September 29, 2003, the contents of which are incorporated herein in their entirety.

  This invention was made in part with government support in grant numbers CA71619 and CA83133 awarded by the National Cancer Institute of the National Institutes of Health. The US government has certain rights in this invention.

BACKGROUND OF THE INVENTION Hematopoietic progenitor cells (such as bone marrow-derived CD34 + stem cells) promote the repair of lesions and damaged tissue and provide promise for the treatment of inherited and acquired human diseases (Asahara et al. (1997) Science 275 , 964-967; Rafii et al. (2003) Nat. Med. 9, 702-12; Takahashi et al., (1999) Nat. Med. 5, 434-438; Kawamoto et al. (2001) Circulation 103, 634-637; Hattori et al. (2001) J. Exp. Med. 193, 1005-1014; Otani et al. (2002) Nat. Med. 8, 1004-1010 (2002); Priller et al. (2001) J. Cell Biol. 155, 733-738; LaBarge et al. (2002) Cell. 111, 589-601; and Torrente et al. (2003) J. Cell Biol. 162, 511-520). For example, bone marrow-derived CD34 + stem cells promote neovascularization by differentiating into endothelial cells (Asahara et al. (1997) supra; Rafii et al. (2003) Nat. Med. 9, 702-12; Takahashi et al. al. (1999) Nat. Med. 5, 434-438; Kawamoto et al. (2001) Circulation 103, 634-637; Hattori et al. (2001) J. Exp. Med. 193, 1005-1014; Otani et al. (2002) Nat. Med. 8, 1004-1010 (2002); Priller et al. (2001) J. Cell Biol. 155, 733-738; LaBarge et al. (2002) Cell. 111, 589-601 ; Torrente et al. (2003) J. Cell Biol. 162, 511-520; Lyden et al. (2001) Nat. Med. 7, 1194-201; Ruzinova et al. (2003) Cancer Cell. 4: 277- 289; Jain et al. (2003) Cancer Cell 3, 515-516; Religa et al. (2002) Transplantation 74, 1310-1315; and Boehm et al. (2004) J. Clin. Invest. 114, 419-426 ). Neovascularization stimulates the healing of damaged tissue (Asahara et al. (1997) supra; Rafii et al. (2003) Nat. Med. 9, 702-12; Takahashi et al. (1999) Nat. Med. 5, 434-438; Kawamoto et al. (2001) Circulation 103, 634-637; Hattori et al. (2001) J. Exp. Med. 193, 1005-1014; Otani et al. (2002) Nat. Med. 8, 1004-1010 (2002); and Carmeliet (2003) Nat. Med. 9, 653-660), but it also promotes undesirable consequences such as tumor growth and inflammatory disease (Lyden et al. al. (2001) Nat. Med. 7, 1194-201; Ruzinova et al. (2003) Cancer Cell. 4: 277-289; Jain et al. (2003) Cancer Cell 3, 515-516; Carmeliet (2003) Nat. Med. 9, 653-660; and Hanahan et al. (1996) Cell 86, 353-364).

  Although the technology recognizes some of the advantages of using hematopoietic progenitor cells, it remains unclear how hematopoietic progenitor cell adhesion, differentiation and migration can be regulated. Accordingly, there remains a need for methods for altering the adhesion and / or migration of hematopoietic progenitor cells to target tissues and for altering the differentiation of hematopoietic progenitor cells into secondary cells.

SUMMARY OF THE INVENTION The present invention provides a method for altering the adhesion and / or migration of hematopoietic progenitor cells (HPC) to a target tissue and for altering the differentiation of hematopoietic progenitor cells into secondary cells. To meet the needs in the art. The invention also provides for screening test compounds for altering the level of adhesion and / or migration of hematopoietic progenitor cells to a target tissue and for altering differentiation of hematopoietic progenitor cells into secondary cells. Provide a method. The invention further provides a method for isolating hematopoietic progenitor cells.

  In a particularly preferred embodiment, the present invention provides a method for altering the level of adhesion of hematopoietic progenitor cells to a target tissue comprising the following steps: a) i) comprising hematopoietic progenitor cells expressing integrin α4β1 Providing a cell population, ii) a target tissue that is not bone marrow endothelial tissue, and iii) one or more agents that alter specific binding of integrin α4β1 to integrin α4β1 ligand, and b) one or more Treating the cell population and target tissue of said agent with an agent under conditions for specific binding of integrin α4β1 to integrin α4β1 ligand. In one embodiment, the processing step further comprises altering the level of transendothelial migration of hematopoietic progenitor cells. In another embodiment, the processing step further comprises altering the level of differentiation of hematopoietic progenitor cells into secondary cells, as compared to adjacent normal tissue and / or other normal organs (e.g. , Examples 22-23 and FIGS. 36a-b). In another embodiment, the processing step does not include changing the level of angiogenesis in the target tissue.

  While not intended to limit the type or source of HPC, in one embodiment, the HPC can be genetically modified or wild type. In another embodiment, the HPC comprises CD34 + non-endothelial cells and / or CD34 + CD133 + cells that can differentiate into endothelium (FIG. 34a). In a further embodiment, HPC is any tissue (lung tissue, breast tissue, prostate tissue, cervical tissue, pancreatic tissue, colon tissue, ovarian tissue, stomach tissue, esophageal tissue, oral tissue, tongue tissue, gingival tissue, skin tissue. , Muscle tissue, heart tissue, liver tissue, bronchial tissue, cartilage tissue, bone tissue, testicular tissue, kidney tissue, endometrial tissue, uterine tissue, bladder tissue, bone marrow tissue, lymphoma tissue, spleen tissue, thymus tissue, thyroid tissue , Hematopoietic stem cells, endothelial progenitor cells contained in brain tissue, neuronal tissue, gallbladder tissue, ocular tissue (e.g. cornea, uveum, choroid, macular, vitreous humor etc.) and joint tissue (e.g. synovial tissue etc.) Lymphoid endothelial progenitor cells, mesenchymal progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, granulocyte progenitor cells, macrophage progenitor cells, megakaryocyte progenitor cells, erythroid progenitor cells, pro-B cells and pro-T cells Including bone marrow progenitor cells, peripheral blood progenitor cells, umbilical cord progenitor cells, one or more of CD34 + progenitor cells. In one embodiment, the bone marrow progenitor cells comprise one or more of CD31 + cells (Example 24, FIGS. 36e-f), cKit + cells, VEGFR1 + cells, VEGFR2 + cells and CD34 + cells.

  The present invention is not intended to be limited to the type or source of target tissue. Nonetheless, in one embodiment, the target tissue is vascular endothelium, muscle, neuron, tumor, inflammatory, peripheral blood, umbilical cord blood, heart, eye, skin, synovial fluid, tumor, lung, breast, prostate, cervix , Pancreas, colon, ovary, stomach, esophagus, oral cavity, tongue, gingiva, skin, liver, bronchi, cartilage, testis, kidney, endometrium, uterus, bladder, spleen, thymus, thyroid, brain, neurons, gallbladder, eye And one or more of the joint tissues. In preferred embodiments, the tissue is damaging, ischemic and / or malignant (such as metastatic malignant tumor tissue). In another embodiment, the target tissue comprises one or more of fibronectin and vascular tissue. In preferred embodiments, the vascular tissue comprises one or more cell types such as endothelial cells, pericytes, vascular smooth muscle, angiogenic tissue, and non-angiogenic tissue.

  The present invention is not intended to be limited to the source or type of secondary cells from which HPC differentiates. In one embodiment, the secondary cell type is mesenchymal, such as, but not limited to, one or more of fibroblasts, myofibroblasts, stromal cells, pericytes, vascular smooth muscle cells, endothelial cells. Contains cell precursors and / or mesenchymal cells. In another embodiment, the secondary cell type is one or more of epidermal cells, secretory cells, hair cells, corneal cells, hepatocytes, alveolar cells, lung cells, skin cells, intestinal cells, and kidney cells. Including epithelial cells. In preferred embodiments, the secretory cell is selected from one or more of breast epithelial cells, intestinal cells and sebaceous epithelial cells, and the hair cells are selected from one or more of ear hair cells and skin hair cells. The In further embodiments, the secondary cell type is a muscle cell precursor, such as, but not limited to, one or more of skeletal muscle cells, cardiac muscle cells, vascular smooth muscle cells, endocardial cells, and cardiomyocytes. Contains body and / or muscle cells. In yet another embodiment, the secondary cell type is, but not limited to, neuronal precursors and / or neurons such as, but not limited to, one or more of astrocytes, Schwann cells, Purkinje cells, dendritic cells and glial cells. Contains cells. In another embodiment, the secondary cell types are B lymphocyte cells, T lymphocyte cells, monocytes / macrophage cells, granulocyte cells, eosinophil cells, neutrophil cells, natural killer cells, and megakaryocyte cells A monocyte cell includes, but is not limited to, one or more of macrophage cells, osteoclasts and osteoblasts, including immune cell precursors and / or immune cells such as one or more of Is done. In yet another embodiment, the secondary cell type comprises embryonic cell precursors, embryonic cells, melanocyte precursors, melanocytes, myoepithelial cell precursors and / or myoepithelial cells, such as those found in glandular tissue. Including.

  The present invention is not limited to the location of treatment with HPC and target tissue agents. In one embodiment, the treatment step can be in vitro (Examples 19, 20), ex vivo, and in vivo in a mammalian subject (Example 21). In a preferred embodiment, the mammalian subject is selected from one or more of having a disease, predisposed to having the disease, suspected of having the disease, and suspected of having the disease. More specifically, the treatment stage is selected from one or more before, during and after the manifestation of one or more symptoms of the disease. In one preferred embodiment, the mammalian subject is a human.

  In one embodiment, the disease is, but is not limited to, neoplasm, diabetic retinopathy, macular degeneration associated with neovascularization, psoriatic hemangioma, gingivitis, rheumatoid arthritis, osteoarthritis, inflammation, and inflammation Angiogenic, such as one or more of genital bowel disease. Although not intended to limit the target tissue in a subject, in one embodiment, the tissue comprises one or more of ocular tissue, skin tissue, bone tissue, and synovial tissue, wherein the ocular tissue comprises the retina, the macula, Illustrated by the cornea, choroid and vitreous humor. In another embodiment, the tissue comprises a tumor such as a malignant tumor, and more preferably a metastatic malignant tumor.

  In another embodiment, the disease is not angiogenic. In some embodiments, but not limited to fibrosis (hematopoietic progenitor cells differentiate into fibroblasts or other cells in exemplary tissues of lung, liver, heart, skin and / or corneal cells. ), Atherosclerosis (hematopoietic progenitor cells differentiate into exemplary macrophages / monocytes, vascular smooth muscle cells, and / or endothelial cells in the vessel wall), restenosis (hematopoietic progenitor cells are vascular smooth Chronic inflammatory diseases such as myocytes, monocytes / macrophages, eosinophils, granulocytes and / or other immune cells in the vessel wall), rheumatoid arthritis (hematopoietic progenitor cells are endothelial Synovial cells that digest cells, pericytes and / or cartilage, differentiate into monocytes / macrophages that secrete angiogenic and inflammatory factors), asthma (hematopoietic progenitors, eosinophils and their immune cells) , Endothelial cells, pericytes and / or fibroblasts), metastatic cancers (hematopoietic progenitor cells are malignant cells and / or fibroblasts, endothelial cells, smooth muscle cells) Such as stromal cells and / or monocytes), myocardial infarction (hematopoietic progenitor cells differentiate into inflammatory cells arising from hematopoietic stem cells and / or fibroblasts arising from hematopoietic stem cells), and hemorrhagic stroke To the target tissue in non-angiogenic diseases such as in (brain), acute respiratory diseases, myocardial infarction, peripheral arterial injury (suppresses inflammatory cells arising from hematopoietic stem cells), ischemic diseases It may be desirable to reduce the adhesion of HPC.

  In another embodiment, it may be desirable to increase adhesion of HPC to the target tissue in non-angiogenic diseases such as in subjects who have undergone bone marrow transplant and subjects who will receive bone marrow transplant, Selected from one or more before, during and after transplantation. In another embodiment, the mammalian subject is selected from a subject that has undergone hematopoietic progenitor cell transplantation and a subject that will undergo hematopoietic progenitor cell transplantation, and the treatment stage is one before, during, and after hematopoietic progenitor cell transplantation. Selected from one or more. In yet further embodiments, the mammalian subject has a wound and / or is susceptible to developing tissue (including but not limited to burns, skin wounds, cosmetic surgery and internal surgery) to any tissue and organ. Surgical wounds, scar replacement, myocardial infarction (the invention is useful for repairing tissue by stimulating epithelial tissue repair and cardiomyocyte development by augmenting blood vessels, regeneration), severed nerves (e.g., nerve Cells and any type of endothelial cells), damaged brain (including nerve cells and endothelial cells), damaged muscle (including muscle cells and endothelial cells), muscular dystrophy-Duchenne and skeletal muscle cells Other diseases, including peripheral arterial ischemic injury (PAD) (the present invention includes homing and adhesion by hematopoietic progenitor cells, as well as muscle cells, neurons, endothelial cells, pericytes Useful for stimulating the differentiation of hematopoietic progenitors into vesicles and / or vascular smooth muscle), stroke (the present invention provides homing, adhesion and hematopoiesis into neurons and / or vascular cells Useful for stimulating the differentiation of progenitor cells), Parkinson's disease (the present invention is useful for stimulating homing, adhesion, and differentiation of hematopoietic progenitors into cells that produce serotonin All types of wound healing including congenitally damaged muscles such as in). In another embodiment, the mammalian subject has diabetes and / or is susceptible to developing diabetes. Useful for stimulating differentiation). In further embodiments, the mammalian subject has AIDS and / or is susceptible to developing (the present invention is directed to hematopoietic progenitor T cells that can stimulate homing, adhesion, and tissue T cell repopulation with hematopoietic progenitors. Useful for stimulating differentiation). In another embodiment, the mammalian subject has cancer and / or is susceptible to developing (the invention relates to homing, adhesion, and anti-cancer immune cells such as T cells and natural killer cells with hematopoietic progenitor cells. Useful for stimulating the differentiation of hematopoietic progenitor cells).

  The present invention is not intended to be limited to a particular type or source of agent that alters adhesion and / or migration of HPC to a target tissue and alters differentiation of HPC into secondary cell types. In one embodiment, the agent is an antibody exemplified by an antibody including, but not limited to, an anti-integrin α4β1 antibody (eg, Examples 19-21, FIGS. 34b, de, and 35b-d). Including peptides. In one embodiment, the specificity of binding of the anti-integrin α4β1 antibody comprises anti-β2 integrin antibody (Example 24), cIgG antibody (Example 24), anti-αvβ5 (P1F6), and α5β1 (P1F6) (FIGS. 17-20). And 34) and anti-αvβ3 (LM609) (FIG. 20). In another embodiment, the agent comprises one or more of an anti-vascular cell adhesion molecule antibody and an anti-fibronectin antibody. In another embodiment, the agent comprises an antisense sequence such as, but not limited to, an antisense sequence comprising one or more of integrin α4β1 antisense sequence, vascular cell adhesion molecule antisense sequence, and fibronectin antisense sequence. Including. In yet another embodiment, the agent comprises a ribozyme such as, but not limited to, an integrin α4β1 ribozyme, a vascular cell adhesion molecule ribozyme, and a ribozyme including fibronectin ribozyme. Although the present invention is not limited to the mechanism of action of an agent, in one embodiment, the agent a) induces expression of α4β1 on HPC, b) one or more of its ligands of integrin α4β1 Activating α4β1 on HPC, such as by increasing the level of specific binding to, and c) inducing expression of one or more α4β1 ligands by one or more cell types Can function by one or more.

  It is also not intended that the present invention be limited to any particular type or source of integrin α4β1 ligand. In one preferred embodiment, the ligand comprises one or more of vascular cell adhesion molecule (VCAM) and fibronectin.

  The present invention provides a method for altering the level of transendothelial migration of hematopoietic progenitor cells to a target tissue comprising the following steps: a) a cell population comprising hematopoietic progenitor cells expressing integrin α4β1, ii) providing a target tissue that is not bone marrow endothelial tissue, and iii) one or more agents that alter the specific binding of integrin α4β1 to integrin α4β1 ligand, and b) one of the cell population and target tissue Treating one or more with the agent under conditions for specific binding of integrin α4β1 to integrin α4β1 ligand, thereby altering the level of transendothelial migration of hematopoietic progenitor cells into the target tissue. In one embodiment, the processing step does not include altering the level of angiogenesis in the tissue in which hematopoietic progenitor cells migrate.

  Further provided herein is a method for altering the level of differentiation of hematopoietic progenitor cells into a secondary cell type that is not a bone marrow endothelial cell, comprising the following steps: a) i) expressing integrin α4β1 Providing a cell population comprising hematopoietic progenitor cells, and ii) one or more agents that alter the specific binding of integrin α4β1 to integrin α4β1 ligand, and b) the cell population of integrin α4β1 integrin α4β1 Treating with an agent under conditions for specific binding to a ligand, thereby altering the level of differentiation of hematopoietic progenitor cells into a secondary cell type. In one embodiment, the processing step does not include changing the level of angiogenesis in a tissue in which hematopoietic progenitor cells differentiate.

  The present invention also provides a method for screening test compounds for altering the level of hematopoietic progenitor cell adhesion to a target tissue that is not bone marrow endothelial tissue, comprising the following steps: a) i) integrin α4β1 Ii) providing a second composition comprising one or more integrin α4β1 ligands, and iii) a test compound, b) providing a test compound of the first composition and the second composition Contacting one or more with conditions for specific binding of integrin α4β1 to integrin α4β1 ligand, and c) integrin α4β1 in the presence of the test compound as compared to in the absence of the test compound Detects changes in the level of specific binding to integrin α4β1 ligand, thereby altering the level of adhesion of hematopoietic progenitor cells to the target tissue Identifying a test compound. In one embodiment, the method further comprises identifying a test compound as altering one or more levels of hematopoietic progenitor cell migration and differentiation of hematopoietic progenitor cells into a secondary cell type. Changes in the level of migration and differentiation can be compared to control adjacent normal tissues or to other normal organs (e.g., inhibition of HPC differentiation as illustrated in Example 22, Example 23, Figures 36a-b). Such). The contacting step is not limited to any particular location, but can be in vitro (Examples 19, 20), ex vivo, and in vivo in a non-human mammal (Example 21).

  The present invention further provides a method for screening test compounds for altering the level of transendothelial migration of hematopoietic progenitor cells to a target tissue that is not bone marrow endothelial tissue, comprising the following steps: a) i) Providing a first composition comprising integrin α4β1, ii) a second composition comprising one or more integrin α4β1 ligands, and iii) a test compound, b) a test compound comprising the first composition and the second composition Contacting one or more of the product with conditions for specific binding of integrin α4β1 to integrin α4β1 ligand, and c) in the presence of the test compound as compared to in the absence of the test compound Detects changes in the level of specific binding of integrin α4β1 to integrin α4β1 ligand, thereby causing transendothelial migration to tissue of hematopoietic progenitor cells Identifying a test compound to alter.

  The invention also provides a method for screening a test compound for altering the level of differentiation of hematopoietic progenitor cells into a secondary cell type that is not bone marrow endothelial tissue, comprising the following steps: a) i) providing a first composition comprising integrin α4β1, ii) providing a second composition comprising one or more integrin α4β1 ligands, and iii) a test compound, b) providing the test compound as the first composition and Contacting one or more of the two compositions with integrin α4β1 under conditions for specific binding of integrin α4β1 ligand, and c) presence of the test compound compared to in the absence of the test compound Underlying changes in the level of specific binding of integrin α4β1 to integrin α4β1 ligand, thereby determining the level of differentiation of hematopoietic progenitor cells into secondary cell types Thereby identifying a changing the test compound.

  The present invention further provides a method for isolating hematopoietic progenitor cells from tissue comprising the following steps: a) i) tissue containing hematopoietic progenitor cells, ii) specifically binding to integrin α4β1 polypeptide Providing an antibody; b) treating the tissue with an agent under conditions such that the antibody binds to hematopoietic progenitor cells; and isolating hematopoietic progenitor cells that bind to the antibody.

Definitions In order to facilitate understanding of the present invention, several terms are defined below.

  As used in this specification and the appended claims, the singular forms “a”, “an” and “the” are clearly different in content. Unless stated otherwise, includes both singular and plural references.

  As used herein, the term “or” when used in the phrase “A or B” and A and B refer to a composition, disease, product, etc. means one, the other, or both. .

  The term “on” when referring to the position of a first object relative to a second object is, for example, the second object after the first object is first placed on the second object. It means that the first object, including the case of penetrating the object, is above and / or in the second object.

  As used herein, the term “comprising” when placed before a list of steps in a method, includes that the method includes one or more steps that are in addition to those explicitly listed; and It means that one or more additional steps can be performed before, during and / or after the listed steps. For example, the method comprising steps a, b and c comprises the method of steps a, b, x and c and the method of steps a, b, c and x, in addition to the method of steps x, a, b and c. Including. Furthermore, the term “comprising” when placed before an enumeration of steps in a method does not require sequential execution of the listed steps unless the content clearly dictates otherwise. In some cases). For example, a method comprising steps a, b and c includes a method of performing steps in the order of steps a, c and b, steps c, b and a, steps c, a and b, etc. Including.

  Unless otherwise specified, as used in the specification and claims, all numbers representing amounts of ingredients, properties such as molecular weight, reaction conditions, etc. are modified by the term “about” in all examples. Should be understood as being. Accordingly, unless specified to the contrary, the numerical parameters in the specification and claims are approximations that can vary depending on the desired properties sought to be obtained by the present invention. At a minimum, and without limiting the application of the principle of equivalence to the scope of the claims, each numeric parameter takes into account the number of reported significant digits and by applying normal rounding techniques. Should be interpreted at least. Although the numerical ranges and parameters describing the broad scope of the invention are approximations, the numerical values in the specific examples are reported as accurately as possible. Any numerical value, however, inherently contains standard deviations necessarily resulting from the errors found in the numerical testing measurements.

  Placed before any particularly specified molecule (such as protein, nucleotide sequence, etc.) or phenomenon (such as cell adhesion, cell migration, cell differentiation, angiogenesis, biological activity, biochemical activity, etc.), and The term “not” when referred to in that sense means that only the specified molecule or phenomenon is specifically excluded.

  As used herein with respect to the level of any molecule (such as protein, nucleotide sequence, etc.) or phenomenon (such as cell adhesion, cell migration, cell differentiation, angiogenesis, biological activity, biochemical activity, etc.) The terms “changing” and grammatical equivalents refer to an increase and / or decrease in the amount of a substance and / or phenomenon, regardless of whether the amount is measured objectively and / or subjectively.

  Regarding the level of molecules (such as proteins, nucleotide sequences, etc.) or phenomena (such as cell adhesion, cell migration, cell differentiation, angiogenesis, biological activity, biochemical activity, etc.) in the first sample relative to the second sample The terms “increase”, “elevate”, “elevate” and grammatical equivalents of any technique such as Student's t-test in which the amount of substance and / or phenomenon in the first sample is greater than in the second sample. Means any amount that is statistically significant using a field-accepted statistical analysis method. In one embodiment, the increase can be measured subjectively if, for example, the patient refers to their subjective perception of disease symptoms such as pain, visual clarity, and the like. In another embodiment, the amount of substance and / or phenomenon in the first sample is at least 10%, preferably at least 25%, more preferably at least 50% greater than the amount of the same substance and / or phenomenon in the second sample. % More, even more preferably at least 75% more, and most preferably at least 90% more.

  Relates to the level of molecules (such as proteins, nucleotide sequences, etc.) or phenomena (such as cell adhesion, cell migration, cell differentiation, angiogenesis, biological activity, biochemical activity, etc.) in the first sample relative to the second sample The terms “reduce”, “inhibit”, “reduce”, “suppress”, “reduce” and grammatical equivalents are used to describe the amount of substance and / or phenomenon in a first sample Means lower by any amount that is statistically significant using any technically accepted statistical analysis method. In one embodiment, the reduction can be measured subjectively if, for example, the patient refers to their subjective perception of disease symptoms such as pain, visual clarity and the like. In another embodiment, the amount of substance and / or phenomenon in the first sample is at least 10% less, preferably at least 25% less, more preferably at least 50 than the amount of the same substance and / or phenomenon in the second sample. % Less, even more preferably at least 75% less, and most preferably at least 90% less. A decrease in the level of a molecule and / or phenomenon may mean an absolute absence of the molecule and / or phenomenon, but it is not necessary.

  Reference herein to any specifically designated protein (such as `` integrin α4β1 '', `` vascular cell adhesion molecule '', fibronectin, etc.) refers to the biological activity of the specifically designated protein (this A polypeptide having at least one of those disclosed in the specification and / or known in the art, and biological activity can be detected by any method. In preferred embodiments, the amino acid sequence of the polypeptide has at least 95% homology (ie, identity) with the amino acid sequence of the specifically designated protein. References herein to any specifically designated protein (such as “integrin α4β1”, “vascular cell adhesion molecule”, fibronectin, etc.) also refer to the amino acid sequence of the specifically designated protein. Specifically included within the scope are specifically designated protein fragments, fusion proteins, and variants having at least 95% homology.

  The term “fragment” when referring to a protein refers to the portion of the protein that can range in size from four consecutive amino acid residues to the entire amino acid sequence minus one amino acid residue. Thus, a polypeptide sequence comprising “at least a portion of an amino acid sequence” comprises from 4 consecutive amino acid residues to the entire amino acid sequence of the amino acid sequence.

  As used herein, the term “variant” of a protein is defined as an amino acid sequence that differs from that protein by one or more amino acid insertions, deletions, and / or conservative substitutions. The term “conservative substitution” of an amino acid refers to the replacement of that amino acid with another amino acid having similar hydrophobicity, polarity and / or structure. For example, the following aliphatic amino acids with neutral side chains can be used conservatively instead of one another: glycine, alanine, valine, leucine, isoleucine, serine and threonine. Aromatic amino acids with neutral side chains that can be used conservatively in place of one another include phenylalanine, tyrosine, and tryptophan. Cysteine and methionine are sulfur-containing amino acids that can be used conservatively instead of one another. Asparagine can also be used conservatively instead of glutamine, and vice versa, since both amino acids are amides of the amino acids of dicarboxylic acids. Furthermore, aspartic acid can be used conservatively instead of glutamic acid, since both are acidic charged (hydrophilic) amino acids. Also, lysine, arginine and histidine can be used conservatively instead of one another because each is a basic charged (hydrophilic) amino acid. Guidance in determining which amino acid residues and how many amino acid residues can be substituted, inserted or deleted without loss of biological and / or immunological activity is well known in the art Computer programs such as DNAStar ™ software. In one embodiment, the variant sequence has at least 95% identity, preferably at least 90% identity, more preferably at least 85% identity, even more preferably at least 75% identity with the sequence of the protein in question. Even more preferably at least 70% identity, and even more preferably at least 65% identity.

  Reference herein to any specifically designated nucleotide sequence (such as the sequence encoding integrin α4β1) includes fragments, homologues of specifically designated nucleotide sequences, and specifically Hybridizes to a specified nucleotide sequence under conditions of high and / or moderate stringency and the biological activity of the specifically specified nucleotide sequence (as disclosed herein and / or in the art) Within that range is a sequence having at least one of those (such as those known in the art) and the biological activity can be detected by any method.

  Nucleotide “fragments” can range in size, illustratively from 10, 20, 50, 100 contiguous nucleotide residues to the entire nucleic acid sequence minus one nucleic acid residue. Thus, a nucleic acid sequence comprising “at least a portion” of a nucleotide sequence includes from 10 consecutive nucleotide residues to the entire nucleotide sequence of the nucleotide sequence.

  The term “homolog” of a specifically designated nucleotide sequence refers to at least 95% identity, more preferably at least 90% identity, even more preferably at least 85% identity, with the sequence of the nucleotide sequence in question. More preferably, it refers to an oligonucleotide sequence having at least 80% identity, more preferably at least 75% identity, even more preferably at least 70% identity, and most preferably at least 65% identity.

  For sequences that hybridize under stringent conditions to a specifically designated nucleotide sequence, high stringency conditions are 5X SSPE, 1% SDS, 5X when probes of about 100 to about 1000 nucleotides in length are used. Equivalent to specific binding or hybridization at 68 ° C in a solution containing Denhardt's reagent and 100 μg / ml denatured salmon sperm DNA, followed by a wash at 68 ° C in a solution containing 0.1X SSPE and 0.1% SDS Includes conditions. `` Medium stringency conditions '' when used for nucleic acid hybridization are 5X SSPE (43.8 g / l NaCl, 6.9 g / l NaH2PO4-H2O and 1.85 g / l EDTA, pH adjusted to 7.4 with NaOH), binding or hybridization at 42 ° C in a solution of 0.5% SDS, 5X Denhardt's reagent and 100 μg / ml denatured salmon sperm DNA, followed by 1.0X SSPE, 1.0% Includes conditions equivalent to washing at 42 ° C. in a solution containing SDS.

  The term `` equivalent '' when it is described with respect to hybridization conditions when it is related to the hybridization conditions of interest means that the hybridization conditions and the hybridization conditions of interest result in the same range of percentages (% Means that hybridization of homologous nucleic acid sequences occurs. For example, if the hybridization condition of interest results in hybridization of the first nucleic acid sequence with another nucleic acid sequence having 85% to 95% homology with the first nucleic acid sequence, another If one hybridization condition also results in hybridization of the first nucleic acid sequence with another nucleic acid sequence having 85% to 95% homology with the first nucleic acid sequence, It is said to be equivalent to the subject hybridization conditions.

  As will be appreciated by those skilled in the art, it may be advantageous to generate a nucleotide sequence encoding a protein of interest having non-naturally occurring codons. Therefore, in some preferred embodiments, the codons preferred by certain prokaryotic or eukaryotic hosts (Murray et al., Nucl. Acids Res., 17 (1989)) are, for example, naturally occurring sequences. Is selected to increase expression rate or to produce a recombinant RNA transcript having desirable properties such as a longer half-life than the transcript produced from

  The term `` naturally occurring '' as used herein when applied to an object (such as a cell) and / or chemical (such as an amino acid, amino acid sequence, nucleic acid, nucleic acid sequence, codon, etc.) "Means that the object and / or compound can be found in nature. For example, a naturally occurring polypeptide sequence refers to a polypeptide sequence present in an organism (including viruses) that can be isolated from a natural source, which polypeptide sequence has been deliberately modified by humans in the laboratory. Not.

  The terms nucleotide sequence “including a specific nucleic acid sequence” and protein “including a specific amino acid sequence” and the equivalents of these terms include a specifically designated nucleic acid sequence and a specifically designated amino acid sequence, respectively. To any nucleotide sequence and any protein of interest. The present invention relates to the source (e.g., cell type, tissue, animal, etc.), nature (e.g., synthetic, recombinant, purified from cell extract, etc.) of the nucleotide sequence of interest and / or the protein of interest, And / or not limited to sequences. In one embodiment, the nucleotide sequence of interest and the protein of interest include coding sequences for structural genes (eg, probe genes, reporter genes, selectable marker genes, oncogenes, drug resistance genes, growth factors, etc.).

  The term “selected from A, B and C” means selecting one or more of A, B and C.

  As used herein, “composition comprising a particular polynucleotide sequence” refers broadly to any composition comprising a read-out polynucleotide sequence. The composition can include, for example, an aqueous solution containing a salt (eg, NaCl), a surfactant (eg, SDS), and other components (eg, Denhardt's solution, dry milk, salmon sperm DNA, etc.).

  The terms “hematopoietic progenitor cells” and “HPC” refer to cells that are not committed (ie, undifferentiated) and / or partially committed (ie, partially differentiated). Hematopoietic progenitor cells are oligopotent, ie, they are not limited to granulocytes (eg, promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (eg, reticulated). It has the ability to differentiate into more than one cell type including red blood cells, red blood cells), platelets (eg megakaryblasts, platelet producing megakaryocytes, platelets), and monocytes (eg monocytes, macrophages).

  Hematopoietic progenitor cells are usually but not always present in the bone marrow. They are also found in the blood circulation and are present in other tissues. Hematopoietic progenitor cells are identified by surface markers. For example, human progenitor cells are identified by the surface marker CD34 (CD34 + cells). For example, 0.1% of circulating cells in the blood are CD34 + and 2.1% of bone marrow cells are CD34 +. Hematopoietic stem cells present in the tissue were also found to be CD34 +. Bone marrow derived (i.e., isolated from bone marrow or isolated from circulation) and tissue derived CD34 + cells can differentiate into muscle, neural tissue, epithelial tissue, vascular cells, immune cells, etc. and repopulate the target tissue Can be used for Hematopoietic progenitor cells have been used therapeutically in vivo to repopulate damaged and diseased tissues and participate spontaneously in tissue repair processes and pathology (Belicci et al., (2004) J. Neurosci Res 77, 475-86; Otani et al., 2002, Nature Med. 8, 1004-1010; Otani et al., (2004) J. Clin. Invest. 114, 765-774; Tamaki et al., (2002 ) J. Cell Biol. 157, 571-577; Torrente et al., (2004) J. Clin. Invest. 114, 182-195; Hashimoto et al., (2004) J. Clin. Invest. 113, 243- 252).

  The term “hematopoietic progenitor cells” clearly includes hematopoietic stem cells, endothelial progenitor cells, lymphoid endothelial progenitor cells, mesenchymal progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, granulocyte progenitor cells, macrophage progenitor cells, megakaryocytes Includes progenitor cells, erythroid progenitor cells, pro-B cells and pro-T cells (Terskikh (2003) Blood 102, 94-101).

  Hematopoietic progenitor cells can be isolated and cultured using the methods disclosed herein and methods known in the art, such as from blood products (e.g., U.S. Patent No. 5,061,620, incorporated by reference). And No. 6,645,489). A “blood product” as used in the present invention defines a product obtained from the body or organ of the body containing cells of the hematopoietic system. Such sources include unfractionated bone marrow, umbilical cord, peripheral blood, liver, thymus, lymph and spleen. It will be apparent to those skilled in the art that all of the aforementioned unpurified or unfractionated blood products can be enriched for cells having “hematopoietic progenitor cell” properties in a number of ways. For example, blood products can be removed from more differentiated offspring. More mature and differentiated cells can be selected on the contrary by the cell surface molecules they express. In addition, blood products can be fractionated to select for CD34.sup. + Cells. Such selection can be achieved using the methods disclosed herein and commercially available magnetic anti-CD34 beads (Dynal, Lake Success, N.Y.). Unfractionated blood products can be obtained directly from the donor or can be recovered from cryopreserved storage.

  The terms “hematopoietic stem cell” and “HSC” include, but are not limited to, endothelial progenitor cells, lymphoid endothelial progenitor cells, mesenchymal progenitor cells, myeloid progenitor cells, lymphoid progenitor cells, granulocyte progenitor cells, macrophage progenitor cells, megakaryocytes Refers to oligo-differentiated cell types that give rise to more differentiated progenitor cells such as sphere progenitor cells, erythroid progenitor cells, pro-B cells and pro-T cells (Terskikh (2003) supra). HSCs often attach to bone and are present in the bone marrow, but are also found in the circulation and are present in other tissues. Hematopoietic stem cells are capable of self-renewal but have no more committed precursors (Terskikh (2003) supra). HSC and HPC have a common cell surface marker, especially for human cells, with the marker CD34. Lineage negative (lack of specific markers for any differentiated cells such as B220 on B cells, CD3 on T cells, CD11b on myeloid cells), CD34 +, c-kit + (Belicci et al. (2004) supra). In mice, these cells are c-kit +, Thy1.1lo, Sca-1 + and Lin- (Rafii et al. 2003, supra). In addition, some precursors, including endothelial precursors, express CD133.

  The terms “endothelial progenitor cell”, “EPC”, “endothelial cell progenitor” and “lymph endothelial progenitor cell” refer to cells that originate from HSC and give rise to differentiated endothelial cells and lymph endothelial cells, respectively. EPCs are CD34 +, CD133 +, c-kit +, and Lin- (Rafii et al. 2003, supra). In addition, it may be VEGFR2 + and / or VEGFR3 + (Rafii et al. 2003, supra). Human endothelial progenitor cells express the surface molecules CD34, flk-1, and / or tie-2 (Isner et al., US Pat. No. 5,980,887, the entire contents of which are hereby incorporated by reference). Mouse endothelial cell precursors express the TM gene, tie-2 gene and / or fgf3 gene and / or stain with GSL I B4 lectin (Hatzopoulos et al. (1998) Development 125: 1457-1468).

  The term “mesenchymal progenitor cells” refers to cells that originate from HSC and give rise to fibroblasts and other stromal cells such as bone, adipose tissue and cartilage (Gronthos et al., 2003, J. Cell Sci. 116, 1827-1835).

  The term “myeloid progenitor cell” is a precursor that arises from HSC and produces granulocytes, macrophages, erythrocytes, megakaryocytes (and thus platelets), and optionally endothelial cells, muscle cells, and other tissues. (Terskikh et al. (2003) supra).

  The term “lymphoid progenitor cell” refers to a cell originating from HSC and giving rise to T and B cells (Otani et al. (2002) supra).

  As used herein, the term “tissue exhibiting angiogenesis” refers to tissue in which new blood vessels arise from pre-existing blood vessels.

  As used herein, the terms “inhibiting angiogenesis”, “decreasing angiogenesis”, “decreasing angiogenesis” and their grammatical equivalents are more than the amount in a control tissue. Angiogenesis in tissue, preferably in an amount that is 10% less, more preferably 50% less, even more preferably 75% less, even more preferably 90% less, and most preferably at the same level observed in control tissues It means to lower the level of. A decrease in the level of angiogenesis may mean an absolute absence of angiogenesis, but it need not be. The present invention does not require and is not limited to a method of completely eliminating angiogenesis.

  The level of angiogenesis is measured using methods well known in the art, including, but not limited to, counting the number of blood vessels and / or the number of blood vessel bifurcations, as discussed herein. Can be done. Alternative assays include in vitro cell adhesion assays that show whether a compound inhibits the ability of α4β1-expressing cells (eg, M21 melanoma cells) to adhere to VCAM or fibronectin. Another in vitro assay that is contemplated includes a tubular banding assay that exhibits new blood vessel growth at the cellular level (D.S. Grant et al., Cell, 58: 933-943 (1989)). Technically accepted in vivo assays are also known and include the use of various test animals such as chickens, rats, mice, rabbits and the like. These in vivo assays include the chick chorioallantoic membrane (CAM) assay, which is suitable for exhibiting anti-angiogenic activity in both normal and neoplastic tissues (DH Ausprunk, Amer. J. Path., 79, No. 3: 597-610 (1975) and L. Ossonowski and E. Reich, Cancer Res., 30: 2300-2309 (1980)). Other in vivo assays reduce the growth rate of transplanted tumors in certain mice or suppress the formation of tumor or precancerous cells in mice that are susceptible to cancer or express chemically induced cancers , Including mouse metastasis assays demonstrating compound potency (MJ Humphries et al., Science, 233: 467-470 (1986) and MJ Humphries et al., J. Clin. Invest., 81: 782-790 (1988) )).

  The term “integrin α4β1” is used interchangeably with the terms “CD49d / CD29”, “latest antigen 4”, and “VLA4” to refer to members of the integrin family. An “integrin” is an extracellular receptor that is expressed in a wide variety of cells and binds to a specific ligand in the extracellular matrix. The specific ligand bound by the integrin can include an arginine-glycine-aspartic acid tripeptide (Arg-Gly-Asp; RGD), or a leucine-aspartic acid-valine (Leu-Asp-Val) tripeptide, For example, fibronectin, vitronectin, osteopontin, tenascin, and von Willebrand factor. Integrin α4β1 is a heterodimeric cell surface adhesion receptor composed of α4 and β1 subunits that bind to ligands present in the extracellular matrix (ECM) and on the cell surface. An exemplary α4 polypeptide sequence is shown in FIG. 1, and an exemplary β1 polypeptide sequence is shown in FIG.

  The term “integrin α4β1” is also intended to include a portion of α4β1. The term “portion” when used in reference to a protein (as in “portion of α4β1”) refers to a fragment of the protein. Fragments can range in size from three consecutive amino acid residues to the entire amino acid sequence minus one amino acid residue. Thus, a polypeptide sequence comprising “at least part of an amino acid sequence” comprises from 3 consecutive amino acid residues to the entire amino acid sequence of the amino acid sequence.

(アミノ酸141位からアミノ酸301位まで)を含む。より好ましい態様において、インテグリンα4β1の部分は、配列

(アミノ酸145位からアミノ酸164位まで)、配列

(アミノ酸184位からアミノ酸197位まで)、配列

(アミノ酸219位からアミノ酸224位まで)、配列

(アミノ酸270位からアミノ酸280位まで)、および配列

(アミノ酸34位からアミノ酸85位まで)を含む。代替の態様において、本発明は、明確には、前記の部分を含むα4ポリペプチド配列(図1の配列により例示されている)の部分を含む。そのような配列は、例えば、

を含む。 In one preferred embodiment, the portion of integrin α4β1 comprises a portion of the α4 polypeptide sequence. In a more preferred embodiment, the portion of the α4 polypeptide sequence shown in FIG.

(From amino acid position 141 to amino acid position 301). In a more preferred embodiment, the portion of integrin α4β1 has the sequence

(From amino acid position 145 to amino acid position 164), sequence

(From amino acid position 184 to amino acid position 197), sequence

(Amino acid 219 to amino acid 224), sequence

(From amino acid position 270 to amino acid position 280), and sequence

(From amino acid position 34 to amino acid position 85). In an alternative embodiment, the present invention specifically includes a portion of an α4 polypeptide sequence (illustrated by the sequence of FIG. 1) that includes the aforementioned portion. Such a sequence is, for example,

including.

  The terms `` isolated '', `` purified '' and their grammatical equivalents when used with respect to molecules (e.g., proteins, DNA, RNA, etc.) or objects (e.g., hematopoietic progenitor cells) in a sample are Reduction in the amount of at least one contaminant molecule and / or product from (at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 75%, and most preferably at least 90%) Point to. Thus, purification results in “concentration” or increase in the amount of the desired molecule and / or product relative to one or more other molecules and / or products in the sample.

  “Non-endothelial cells” include, but are not limited to, stem cells, lymphocytes, mesenchymal cells, myeloid cells, lymphoid cells, granulocyte cells, macrophage cells, megakaryocyte cells, erythroid cells, B cells, T Any cell type other than endothelial cells such as cells, bone marrow cells, muscle cells, nerve cells, etc. (ie not an endothelial cell).

  The terms `` disease '' and `` pathological condition '' interrupt or alter the execution of normal function, and environmental factors (such as malnutrition, industrial disasters, or climate), certain infectious agents (parasites, bacteria Or a living animal that may be a response to a congenital defect in an organism (such as various genetic abnormalities), or a combination of these and other factors. Used interchangeably to refer to a condition, sign and / or symptom associated with any damage, interruption, cessation, or disorder of the normal state of either an organ or tissue. The term `` disease '' includes a response to injury, particularly when such a response is excessive, produces symptoms that excessively interfere with the normal activity of the individual, and / or when the tissue does not heal normally ( Excessive is characterized as the degree of interference or the length of interference).

DESCRIPTION OF THE INVENTION The present invention provides a method for altering the adhesion and / or migration of hematopoietic progenitor cells to a target tissue and for altering the differentiation of hematopoietic progenitor cells into secondary cell types, Meet the need in the art. The invention also provides for screening test compounds for altering the level of adhesion and / or migration of hematopoietic cells to a target tissue, and altering differentiation of hematopoietic progenitor cells into secondary cell types. Provide a method. The invention further provides a method for isolating hematopoietic progenitor cells. The methods of the invention are useful, for example, in the diagnosis, prevention, and reduction of symptoms associated with diseases and conditions associated with HPC adhesion, migration and differentiation. The methods of the invention are also useful in isolating HPC cells and determining the mechanisms underlying development and wound healing. The methods of the present invention are based in part on our unexpected discovery that integrin α4β1 plays a role in HPC adhesion, migration and differentiation.

  Hematopoietic stem cells supply up to 15% of new blood vessels in tumors by differentiating into endothelial cells (EC) (Ruzinova et al. Cancer Cell 4: 277-289 (2003)), but some hematopoietic stem cells also Angiogenesis can be promoted by differentiating into cells such as monocytes that secrete angiogenic factors (Cursiefen et al. (2004) J. Clin. Invest. 113: 1040-1050). Since most CD34 + cells expressed the integrin α4β1 and α4β1 antagonists almost completely blocked hematopoietic stem cell homing, the data herein (Examples 17-24) show that α4β1 It shows that both roles are controlled. It is also unclear whether hematopoietic stem cells, partially committed progenitor cells, or a combination of the two are involved in angiogenesis. Our data indicate that endothelial progenitor cells also home to tumors in an α4β1-dependent manner. The data herein thus show that inhibition of α4β1 blocks exemplary hematopoietic stem cell homing to new vasculature and subsequent growth to endothelial cells.

  Exemplary circulating hematopoietic stem cells home to the site of neovascularization (Asahara et al. (1997) supra; Rafii et al. (2003) Nat. Med. 9, 702-12; Takahashi et al. (1999) Nat. Med. 5, 434-438; Kawamoto et al. (2001) Circulation 103, 634-637; Hattori et al. (2001) J. Exp. Med. 193, 1005-1014; Lyden et al. (2001) Nat. Med. 7, 1194-201; Ruzinova et al. (2003) Cancer Cell. 4: 277-289; Jain et al. (2003) Cancer Cell 3, 515-516; Religa et al. (2002) Transplantation 74 , 1310-1315; and Boehm et al. (2004) J. Clin. Invest. 114, 419-426)), which give rise to about 15% of the vasculature (Ruzinova et al. (2003) Cancer Cell. 4: 277-289) is the inventors' consideration. They are also involved in tissue regeneration or pathogenesis by homing to muscle, brain and other tissues and differentiating into muscle, nerve and other cell types (Priller et al. (2001) J. Cell Biol. 155, 733-738; LaBarge et al. (2002) Cell. 111, 589-601; Torrente et al. (2003) J. Cell Biol. 162, 511-520; 13. Religa et al. (2002) Transplantation 74, 1310 -1315; and Boehm et al. (2004) J. Clin. Invest. 114, 419-426).

  The integrin α4β1-VCAM interaction promotes heterotypic cell adhesion during many processes in vivo. α4β1-VCAM interaction is the result of fusion of the chorionic membrane with the allantoic membrane of either molecule (Yang et al. (1995) Development 121, 549-560; and Kwee et al. (1995) Development 121, 489- 503) and fusion of the endocardium with myocardium (Yang et al. (1995) Development 121, 549-560; and Kwee et al. (1995) Development 121, 489-503) Is involved in. The interaction of integrin α4β1 with fibronectin and / or VCAM is also responsible for the adhesion of immune cell precursors to bone marrow ECs and the homing of these cells back to the bone marrow (Simmons et al. (1992) Blood 80, 388- 395; Papayannopoulou et al. (2001) Blood 98, 2403-2411; Craddock et al. (1997) Blood 90, 4779-4788; and Miyake et al. (1991) J. Cell Biol. 114, 557-565), Inflammation (Guan et al. (1990) Cell 60, 53-61; and Elices et al. (1990) Cell 60, 577-584) and cancer (Melder et al. (1996) Nat Med. 2: 992-997) Is involved in immune cell trafficking. In one embodiment, our data show that the novel function of α4β1 interaction with the exemplary ligands VCAM and fibronectin, ie, endothelial cells of exemplary hematopoietic stem cells during neovascularization and tissue remodeling. To promote meetings with

  The data herein (e.g., Examples 17-24, Figures 21-36) show that integrin α4β1 plays a central role in homing exemplary hematopoietic stem cells to tumors, inflammatory and damaged tissues And manipulation of the expression and / or function of integrin α4β1 and its ligands has been shown to provide a means to regulate pathological processes involving hematopoietic progenitor cells such as hematopoietic stem cells.

  The present invention is further discussed below under the following headings: A) an integrin α4β1 ligand, B) an agent that alters the binding of integrin α4β1 to its ligand, and C) integrin α4β1 during neovascularization. Mediate transport of endothelial progenitor cells, as exemplified by endothelial stem cells, D) alter hematopoietic progenitor cell adhesion, migration and differentiation, E) alter hematopoietic progenitor cell adhesion, migration and differentiation F) detection of hematopoietic progenitor cells expressing integrin α4β1, G) screening of compounds, and H) isolation of hematopoietic progenitor cells.

A. Integrin α4β1 Ligand The methods of the invention employ agents that inhibit the specific binding of integrin α4β1 to one or more of its ligands. The term “ligand” as used herein with respect to the ligand for the integrin α4β1 receptor refers to a molecule and / or portion thereof that specifically binds α4β1. In one embodiment, binding of the ligand causes a specific biological response (eg, hematopoietic progenitor cell adhesion, migration and / or differentiation) and / or signal transduction in the cell. The integrin α4β1 ligand can be present on the cell surface or can be present in the extracellular matrix (ECM).

により例示される。 In one preferred embodiment, the integrin α4β1 ligand present on the cell surface is exemplified by a vascular cell adhesion molecule (VCAM). An example of a VCAM polypeptide sequence is shown in FIG. In another preferred embodiment, the integrin α4β1 ligand is part of VCAM. A preferred portion of VCAM (FIG. 3A, GenBank Accession No. P19320) is the amino acid sequence RTQIDSPLNG (SEQ ID NO: 15) (amino acid position 60 to amino acid position 69); amino acid sequence RTQIDSPLSG (SEQ ID NO: 16) (amino acid 348 And amino acid sequence KLEK (SEQ ID NO: 17) (amino acid 103 to amino acid 106 and amino acid 391 to amino acid 394). Other parts of the VCAM are also contemplated and preferably include one or more of the RTQIDSPLNG (SEQ ID NO: 15), RTQIDSPLSG (SEQ ID NO: 16), KLEK (SEQ ID NO: 17) sequences. These are not limited,

Is exemplified by

を含む。一つのより好ましい態様において、部分は、アミノ酸配列LDV(SEQ ID NO:19)(アミノ酸2011位からアミノ酸2013位まで)を含むCS-1配列を含む。代替の態様において、部分は、アミノ酸配列REDV(SEQ ID NO:20)(アミノ酸2091位からアミノ酸2094位まで)を含むCS-5配列を含む。さらにもう一つの好ましい態様において、部分は、アミノ酸配列IDAPS(SEQ ID NO:21)(アミノ酸1903位からアミノ酸1907位まで)を含む。本発明はさらに、限定されるわけではないが、配列

により例示されているように、前記の配列を含むフィブロネクチンの部分を含む。 In another preferred embodiment, the integrin α4β1 ligand present in the ECM is exemplified by fibronectin. An exemplary polypeptide sequence for fibronectin is shown in FIG. In another preferred embodiment, the integrin α4β1 ligand is part of fibronectin. The preferred portion of fibronectin illustrated in FIG. 4 is a IIICS sequence from amino acid 1982 to amino acid 2111 that encodes two α4β1 binding sites.

including. In one more preferred embodiment, the portion comprises a CS-1 sequence comprising the amino acid sequence LDV (SEQ ID NO: 19) (from amino acid 2011 to amino acid 2013). In an alternative embodiment, the portion comprises a CS-5 sequence comprising the amino acid sequence REDEV (SEQ ID NO: 20) (from amino acid 2091 to amino acid 2094). In yet another preferred embodiment, the portion comprises the amino acid sequence IDAPS (SEQ ID NO: 21) (from amino acid position 1903 to amino acid position 1907). The present invention further includes, but is not limited to, sequences

A portion of fibronectin comprising the sequence described above.

  Integrin α4β1 ligands other than VCAM, fibronectin, and portions thereof are also contemplated as being within the scope of the present invention. These ligands can be determined using routine methods available to those skilled in the art. For example, the presence of antibodies against VCAM, fibronectin and integrin α4β1 create a possible method for isolating other integrin α4β1 and integrin α4β1 ligands. One method utilizes the antibody characteristics known as the idiotype method. Each antibody contains a unique region that is specific for an antigen. This area is called an idiotype. The antibody itself contains antigenic determinants; the idiotype of an antibody is an antigenic determinant that is unique to the molecule. By immunizing organisms with antibodies, “anti-antibodies” that recognize antibodies, including antibodies that recognize idiotypes, can be produced. An antibody that recognizes the idiotype of another antibody is called an anti-idiotype antibody. Some anti-idiotype antibodies are said to mimic the original antigen shape recognized by the antibody and have an “internal image” of the antigen (Kennedy (1986) Sci. Am. 255: 48-56). For example, anti-idiotype antibodies have been successfully produced against anti-ELAM1 antibodies and have been found to recognize ELAM1 ligand, a molecule expressed on the surface of endothelial cells (similar to integrin α4β1). (US Pat. No. 6,252,043, which is incorporated by reference in its entirety).

  When the antigen is a ligand, a particular anti-idiotype can bind to the receptor for that ligand. Some of these have been identified and include anti-idiotypes that bind to insulin, angiotensin II, adenosine I, adrenergic receptors, and rat brain nicotine and opiate receptors (Carlsson and Glad (1989) Bio / Technology 7: 567-73).

B. Agents that Change the Binding of Integrin α4β1 to its Ligands Some preferred methods of the invention alter (ie, increase or decrease) the specific binding of α4β1 to one or more of its ligands. Step) utilizing the active substance. The term “specific binding” as used herein with respect to binding of an agent to either integrin α4β1 or integrin α4β1 ligand is the specific structure on the integrin α4β1 or its ligand, respectively. It depends on the existence of For example, if the agent is specific for epitope `` A '', the presence of a protein comprising labeled `` A '' and epitope A in a reaction involving the agent (i.e. free unlabeled A) is Reduce the amount of labeled A bound to the agent.

  The terms “inhibit specific binding” and “reduce specific binding” when used in reference to the effect of an agent on the specific binding of an integrin α4β1 to an integrin α4β1 ligand When the level of specific binding with the ligand is detected, for example by enzyme linked immunoassay (ELISA), preferably 10% less, more preferably 50% less than the amount of specific binding in the control sample, It means even more preferably 75% less, even more preferably 90% less, and most preferably a decrease in amount, which is the same level observed in control samples. A decrease in the level of specific binding may mean an absolute lack of specific binding, but it is not necessary. The present invention does not require and is not limited to a method that completely removes the specific binding of integrin α4β1 to its ligand.

  The term “antagonist” is used herein to mean a molecule (eg, an antibody) that can inhibit the specific binding of a receptor and its ligand. Anti-α4β1 integrin antibodies are examples of α4β1 antagonists that inhibit the specific binding of α4β1 to fibronectin. An antagonist can act as a competitive or non-competitive inhibitor of α4β1 binding to its ligand.

  The terms `` agent '', `` test agent '', `` test compound '', `` compound '', `` molecule '' and `` test molecule '' are obtained from any source, such as plant, animal and environmental sources, Or any type of molecule (e.g., peptides, nucleic acids, sugars, lipids, organic and inorganic molecules, etc.) prepared by any method (e.g., purification of naturally occurring molecules, chemical synthesis, genetic engineering methods, etc.) ). Agents include both known and potential compounds. As described further below, agents are not limited to antibodies, nucleic acid sequences such as antisense and ribozyme sequences, and compounds generated by chemical libraries, phage libraries, etc. Is exemplified by

  While not intending to limit the present invention to any mechanism and that no understanding of the mechanism is required, the agent may specifically bind the integrin α4β1 receptor to its ligand, For example, by binding to a binding site located on the ligand, thereby inhibiting binding of the integrin α4β1 receptor to that binding site on the ligand, or binding to a site other than the binding site on the ligand, It is contemplated that it can be inhibited by a variety of mechanisms, including by sterically hindering the binding of the integrin α4β1 receptor to the binding site on the ligand. Alternatively, the agent may cause conformational or other changes in the receptor that bind to integrin α4β1 (rather than to integrin α4β1 ligand), thereby inhibiting binding of integrin α4β1 to the ligand.

1. Antibodies In one embodiment, the agent that inhibits specific binding of α4β1 to one or more of its ligands is an antibody. The terms “antibody” and “immunoglobulin” are specific for one or more epitopes elicited in an animal by an immunogen and to the immunogen or, more specifically, contained in the immunogen. Refers to a glycoprotein or part thereof. The term “antibody” specifically includes within its scope antigen-binding fragments of such antibodies, including, for example, Fab, F (ab ′) ₂ , Fd, or Fv fragments of antibodies. The antibodies of the present invention also include chimeric and humanized antibodies. The antibody can be polyclonal or monoclonal. The term “polyclonal antibody” refers to an immunoglobulin produced from more than a single clone of plasma cells; in contrast, a “monoclonal antibody” refers to an immunoglobulin produced from a single clone of plasma cells.

  Antibodies contemplated to be within the scope of the present invention include naturally occurring antibodies and, for example, single chain antibodies, chimeric, bifunctional, and humanized antibodies, in addition to antigen binding fragments thereof. Including non-naturally occurring antibodies. Naturally occurring antibodies can be produced in any species including mouse, rat, rabbit, hamster, human and monkey species using methods known in the art. Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly, or can be produced, for example, as previously described (Huse et al., Science 246: 1275-1281 ( 1989)), can be obtained by screening a combinatorial library consisting of a variable heavy chain and a variable light chain. For example, methods including these for making chimeric, humanized, CDR-grafted, single chain, and bifunctional antibodies are well known to those skilled in the art (Winter and Harris, Immunol. Today 14: 243- 246 (1993); Ward et al., Nature 341: 544-546 (1989); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); and Borrabeck, Antibody Engineering, 2d ed. (Oxford University Press 1995).

As used herein, the term “antibody” when used in reference to anti-integrin antibodies, particularly anti-integrin α4β1 antibodies, specifically binds to one or more epitopes on an α4β1 polypeptide or peptide portion thereof. And an antibody that may or may not include some or all of the RGD binding domain. In one embodiment, the anti-integrin α4β1 antibody, or antigen-binding fragment thereof, is at least about ¹ × 10 ⁵ M ⁻¹ , more preferably at least about ¹ × 10 ⁶ M ⁻¹ , and even more preferably at least about ¹ × 10 ⁷ M ⁻¹ integrin. It has a specific binding activity for α4β1.

  Those skilled in the art know how to make polyclonal and monoclonal antibodies that are specific for a desired polypeptide. For example, monoclonal antibodies can be made by immunizing an animal with the desired antigen, and spleen cells from the immunized animal are generally immortalized by fusion with myeloma cells.

  Immunization with an antigen can be accomplished in the presence or absence of an adjuvant (eg, Freund's adjuvant). Typically, for mice, 10 μg antigen in 50-200 μl adjuvant or aqueous solution is administered per mouse by subcutaneous, intraperitoneal or intramuscular route. Booster immunizations can be given at intervals (eg, 2-8 weeks). The final boost is given about 2-4 days prior to fusion and is generally given in aqueous solution rather than in adjuvant.

  Spleen cells from immunized animals gently release the spleen cells to the medium at room temperature by tearing the spleen through a sterile sieve into the medium or by pressure between the matte ends of two sterile glass microscope slides Can be prepared. Cells are collected by centrifugation (5 min, 400xg), washed and counted.

Spleen cells are fused with myeloma cells to produce a hybridoma cell line. Several mouse myeloma cell lines selected for sensitivity to hypoxanthine-aminopterin-thymidine (HAT) are commercially available, for example, Dulbecco's Modified Eagle Medium (DMEM) containing 10-15% fetal calf serum (Gibco BRL). Fusion of myeloma cells and spleen cells can be accomplished using polyethylene glycol (PEG) or by electrofusion using protocols that are routine in the art. The fused cells are distributed into 96-well plates, followed by selection of the fused cells by 1-2 weeks of culture in 0.1 ml DMEM containing 10-15% fetal calf serum and HAT. The supernatant is screened for antibody production using methods well known in the art. Hybridoma clones from wells containing antibody producing cells are obtained (eg, by limiting dilution). Cloned hybridoma cells (4-5 × 10 ⁶ ) are transplanted intraperitoneally in recipient mice, preferably in the BALB / c genetic background. Serum and ascites are typically collected from mice after 10-14 days.

  The present invention also contemplates humanized antibodies that are specific for integrin α4β1 and / or at least a portion of its ligand. Humanized antibodies can be made using methods known in the art, including those described in US Pat. Nos. 5,545,806; 5,569,825 and 5,625,126, the entire contents of which are incorporated by reference Is incorporated by Such methods include, for example, transgenic non-humans that contain human immunoglobulin chain genes and can express these genes to produce a repertoire of antibodies of various isotypes encoded by the human immunoglobulin genes. Includes animal production.

  In preferred embodiments, the antibody is specific (ie, specifically binds) to integrin α4β1 and / or portions thereof. Although the present invention is illustrated using antibodies against the C-terminus of fibronectin and against integrin α4β1, and with exemplary peptide antagonists against integrin α4β1, the present invention uses these specific agents. It is not limited to. Rather, the present invention specifically includes any agent that inhibits the specific binding of integrin α4β1 to one or more integrin α4β1 ligands. In one preferred embodiment, the anti-integrin α4β1 antibody comprises integrin α4β1 at least 2-fold greater, preferably at least 5-fold greater, more preferably it binds another integrin, such as Vβ3 and / or Vβ5. It binds with an affinity that is at least 10 times greater, and even more preferably at least 100 times greater. Anti-integrin α4β1 antibodies include, but are not limited to, HP2 / 1, HP1 / 3, HP1 / 1, HP1 / 7, HP2 / 4 (Sanchez-Madrid et al. (1986) Eur. J. Immunol. 16, 1342- 1349), ALC1 / 4.1, ALC1 / 5.1 (Munoz et al. (1997) Biochem J., 327, 27-733), 44H6 (Quackenbush et al. (1985) J. Immunol. 134: 1276-1285), P1H4 , P4C2, P4G9 (Wayner et al. (1998) J. Cell Biol. 109: 1321), 9C10 (Kinashi et al. (1994) Blood Cells 20: 25-44), 9F10 (Hemler et al. (1987) J Biol. Chem. 262: 11478), B5G10 (Hemler et al. (1987) J. Biol. Chem. 262, 3300-3309), 15/7 (Yednock et al. (1995) J. Biol. Chem. 270 : 28740-28750), SG / 73 (Miyake et al. (1992) J. Cell Biol., 119, 653-662) and mouse anti-human integrin α4β1 antibodies. Also within the scope of the present invention is a humanized anti-human integrin α4β1 antibody (Tubridy et al. (1999) Neurology 53 (3): 466-, such as “ANTEGREN ™” (also known as natalitumab). 72, Sheremata et al. (1999) Neurology 52: No. 5, March 23 1999 and Lin et al. (1998) Current Opinion in Chemical Biology 2: 453-457), and the contents of which are incorporated by reference, Newman et al. ., Chimeric antibody disclosed by US Pat. No. 5,750,105; rat anti-mouse integrin α4β1 antibody such as PS / 2 (Chisholm et al. (1993) European J. Immunol 23: 682-688); Mouse anti-rat α4β1 antibody (Issekutz (1991) J. Immunol 147: 4178-4184); and rat anti-mouse α4β1 antibody (Holzmann et al. (1989) Cell 56: 37-46) such as R1-2 It is.

  In another preferred embodiment, the antibody is specific for VCAM and / or portions thereof. In a more preferred embodiment, the anti-VCAM antibody inhibits binding of VCAM to α4β1 integrin but not to other integrins. Exemplary antibodies include, for example, 4B2 and 1E10, P1B8, and P3C4 (Needham et al. (1994) Cell Adhes. Commun. 2: 87-99; Dittel et al. (1993) Blood 81: 2272-2282), and Including chimeric antibodies disclosed by Newman et al., US Pat. No. 5,750,105, the contents of which are incorporated by reference.

  In yet another preferred embodiment, the antibody is specific for fibronectin and / or portions thereof. In a more preferred embodiment, the anti-VCAM antibody inhibits binding of VCAM to α4β1 integrin but not to other integrins. Such antibodies include, but are not limited to, antibodies to major and minor integrin α4β1 binding sites in the C-terminal region of fibronectin and antibodies to adjacent heparin binding sites that interfere with binding of integrin α4β1 to fibronectin. Exemplary antibodies include P1F11 and P3D4 (Garcia-Pardo et al. (1992) Biochemical and Biophysical Research Communications 186 (1): 135-42); and antibodies 20E10, 21E5, 9E9, 16E6, 19B7, 26G10, 30B6, 36C9, and 39B6 (Mostafavi-Pour et al. (2001) Matrix Biology 20 (1): 63-73).

2. Peptide In an alternative embodiment, an agent that inhibits specific binding of integrin α4β1 to one or more of its ligands is an exemplary peptide EILDVPST (SEQ ID NO : 22) (Varner WO 03/019136 A3). The term “peptide” as used herein refers to at least two amino acids and amino acid analogs covalently linked by peptide bonds and / or analogs of peptide bonds. The term peptide includes oligomers and polymers of amino acids and / or amino acid analogs. The term peptide also includes molecules generally referred to as peptides, which typically include from about 2 amino acids to about 20 amino acids. The term peptide also includes molecules generally referred to as polypeptides, which typically contain from about 20 to about 50 amino acids. The term peptide also includes molecules generally referred to as proteins, which typically contain from about 50 to about 3000 amino acids. The amino acids of the peptide antagonist can be L-amino acids and / or D-amino acids.

  The term “derivative” or “modified” when referring to a peptide means that the peptide comprises at least one derivative amino acid. Amino acid “derivatives” and “modified” amino acids are chemically modified amino acids. Derivative amino acids can be “biological” or “non-biological” amino acids. Chemical derivatives of one or more amino acid members can be achieved by reaction with functional side chains. Illustrative derivatized molecules are derivatized such that, for example, a free amino group forms an amine hydrochloride, p-toluenesulfonyl group, carboxybenzoxy group, t-butyloxycarbonyl group, chloroacetyl group, and / or formyl group. Including molecules that have been Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters and / or other types of esters, and hydrazides. Free hydroxyl groups can be derivatized to form O-acyl and / or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-Im-benzyl histidine. Also included as chemical derivatives are peptides containing naturally occurring amino acid derivatives of the 20 standard amino acids. For example, 4-hydroxyproline can be replaced with proline; 5-hydroxylysine can be replaced with lysine; 3-methylhistidine can be replaced with histidine; homoserine can be replaced with serine; Can be substituted with lysine. Other included modifications are amino terminal acylation (eg, acylation or thioglycolic acid amidation), terminal carboxylamidation (eg, with ammonia or methylamine), and similar terminal modifications. Terminal modifications are useful, as is well known, to reduce sensitivity by proteinase digestion and therefore to increase the half-life of the peptide in solution, particularly in biological fluids where proteases may be present. Exemplary modified amino acids include, but are not limited to, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, β-aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, piperidine acid, 6-aminocaprone Acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid, N- Ethylglycine, N-ethylasparagine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methylglycine, sarcosine, N-methylisoleucine, N-methylvaline, norvaline, Contains norleucine and ornithine. Derivatives also include peptides that contain one or more additions or deletions as long as the required activity is maintained.

  It is contemplated that the amino acids of the peptide include biological amino acids and non-biological amino acids. The term “biological amino acid” refers to any one of the 20 known encoded amino acids that a cell can introduce into a polypeptide translated from mRNA. The term “non-biological amino acid” refers to an amino acid that is not a biological amino acid. Non-biological amino acids are useful, for example, due to their stereochemistry or their chemical nature. Non-biological amino acids, such as norleucine, have side chains similar in shape to methionine. However, due to the lack of side chain sulfur atoms, norleucine is less sensitive to oxidation than methionine. Other examples of non-biological amino acids include aminobutyric acid, norvaline and allo-isoleucine, including hydrophobic side chains that have different steric properties compared to biological amino acids.

  Peptides that are useful in the present invention can be synthesized by several methods including chemical synthesis and recombinant DNA techniques. Synthetic chemistry techniques such as solid phase Merrifield synthesis are preferred for reasons such as purity, absence of undesirable by-products, and ease of preparation. A summary of available technologies is available for solid phase peptide synthesis, Steward et al., Solid Phase Peptide Synthesis, WH Freeman, Co., San Francisco (1969); Bodanszky, et al., Peptide Synthesis, John Wiley and Sons, 2nd edition (1976); J. Meienhofer, Hormonal Proteins and Peptides, 2:46, Academic Press (1983); Merrifield, Adv. Enzymol. 32: 221-96 (1969); Fields et al., Intl. Peptide Protein Res., 35: 161-214 (1990) and U.S. Pat. No. 4,244,946; and several methods for classical solution synthesis, including Schroder et al., The Peptides, Vol 1, Academic Press (New York) (1965). Found in these references. Protecting groups that can be used in the synthesis are also described in Protective Groups in Organic Chemistry, Plenum Press, New York (1973). The solid phase synthesis method consists of the sequential addition of one or more amino acid residues or appropriately protected amino acid residues to the growing peptide chain. Either the amino group or the carboxyl group of the first amino acid residue is protected by a suitable selectively removable protecting group. Different, selectively removable protecting groups are utilized for amino acids containing reactive side chains such as lysine.

  The resulting linear peptides can then be reacted to form their corresponding cyclic peptides. Methods for cyclizing peptides are described in Zimmer et al., Peptides, 393-394 (1992), ESCOM Science Publishers, B.V., 1993. To cyclize peptides containing two or more cysteines through formation of disulfide bonds, Tam et al., J. Am. Chem. Soc., 113: 6657-6662 (1991); Plaue, Int. J Peptide Protein Res., 35: 510-517 (1990); Atherton, J. Chem. Soc. Trans. 1: 2065 (1985); and B. Kamber et al., Helv. Chim. Acta 63: 899 (1980 ) Is useful. Polypeptide cyclization is a useful modification for creating modified peptides (e.g., peptidomimetics) because of the stable structure formed by cyclization and considering the biological activity observed for cyclic peptides. It is.

  Alternatively, selected compounds of the present invention are produced by expression of recombinant DNA constructs prepared according to well-known methods if the peptides are known. Such production would be desirable to provide large quantities or alternative embodiments of such compounds. Production by recombinant means may be desirable over standard solid phase peptide synthesis for peptides of at least 8 amino acid residues. DNA encoding the desired peptide sequence is preferably prepared using commercially available nucleic acid synthesis methods. After these nucleic acid synthesis methods, the DNA encoding the peptide is isolated in purified form. Methods for constructing expression systems for the production of peptides in recombinant hosts are also generally known in the art. A preferred recombinant expression system when transformed into a compatible host is capable of expressing DNA encoding the peptide. Other preferred methods used to produce the peptides are effective to cause expression of the coding DNA to produce the peptides of the invention and ultimately to recover the peptides from the culture. Culturing the recombinant host under conditions.

Expression can be performed in prokaryotic and eukaryotic hosts. Prokaryotes are most frequently represented by various strains of E. coli. However, other microbial strains may also be used, such as gonococci such as various species of Bacillus subtilis, Pseudomonas, or other species. In such prokaryotic systems, plasmid vectors containing replication sites and control sequences derived from a species compatible with the host are used. For example, the flagship commercial vector for E. coli is pBR322 and its derivatives. Commonly used prokaryotic control sequences, including a promoter for transcription initiation, optionally including an operator, along with a ribosome binding site sequence are β-lactamase (penicillinase) and lactose (lac) promoter systems, tryptophan (trp) promoter systems , and λ- generally include promoter used, such as from P _L promoter and N- gene ribosome binding site. However, any available promoter system that is compatible with prokaryotic expression is suitable for use.

  Useful expression systems in eukaryotic hosts include promoters from appropriate eukaryotic genes. For example, a class of promoters useful in yeast includes promoters for the synthesis of glycolytic enzymes (eg, for 3-phosphoglycerate kinase). Other yeast promoters include those derived from the enolase gene or Leu2 gene obtained from YEp13. Suitable mammalian promoters include early and late promoters from SV40, or other viral promoters such as those from polyoma, adenovirus II, bovine papilloma virus, or avian sarcoma virus. Appropriate viral and mammalian enhancers can also be used. When plant cells are used as an expression system, for example, a nopaline synthesis promoter is suitable.

  Once the expression system has been constructed using well-known restriction and ligation techniques, transformation of the appropriate host cell is done using standard techniques appropriate for such cells. The cells containing the expression system are cultured under conditions suitable for production of the peptide, and the peptide is then recovered and purified.

  In a preferred embodiment, the agent that specifically binds integrin α4β1 is more preferably at least about 2 times greater, more preferably at least about 5 times greater than the peptide to integrin α4β1 relative to another integrin such as αVβ3. Are useful in the methods of the invention when they bind with specificity for integrin α4β1, which is at least about 10-fold greater, and most preferably at least about 100-fold greater. Thus, the various RGD and RLD containing peptides identified based on their relatively high binding affinity for integrin αVβ3 or for αVβ5 (PCT / US94 / 13542) as defined herein Are not considered peptide antagonists of the binding of integrin α4β1 to its ligand.

(Xは任意のアミノ酸または修飾アミノ酸である)、(X)C^＊(X)PC^＊(SEQ ID NO:31)(Xは任意のアミノ酸である)、RC^＊DPC^＊(SEQ ID NO:32)、C^＊WLDVC^＊(SEQ ID NO:33)、YC^＊APC^＊(SEQ ID NO:34)およびYC^＊DPC^＊(SEQ ID NO:35)、およびフェニルアシル-C^＊DfC^＊(SEQ ID NO:36)(「f」はD-Pheである)(Jackson et al., J. Med. Chem. (1997) 40(21):3359-68)；RC^＊D(ThioP)C^＊(SEQ ID NO:37)(Nowlin et al., J. Biol. Chem. (1993) Sep 25, 268(27):20352-9)；9-フルオレンカルボキシルRC^＊D(ThioP)C^＊(SEQ ID NO:38)(Cardarelli et al., J. Biol. Chem. (1994) 269(28):18668-73)；EGYYGNYGVYA(SEQ ID NO:39)およびC^＊YYGNC^＊(SEQ ID NO:97)(^＊は環化点を示す)；ならびにそれらの修飾体(Thorsett et al., Inhibitors of leukocyte adhesion (1996) WO9602644)；1-アダマンタンアセチル-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys(SEQ ID NO:41)(Cardarelli et al., J. Biol. Chem. (1994) 269(28):18668-73)を含む。他の例示的なペプチドは、限定されるわけではないが、カーペットバイパー(Echis carinatus)由来のEC3、EC3のサブユニットであり、かつ配列

を有するEC3B、EC3のペプチド断片；およびそれらの修飾体(Brando et al., Biochem. Biophys. Res. Commun. (2000) 267(1):413-417, およびMarcinkiewicz et al., J. Biol. Chem. (1999) 274(18):1 2468-73)；可溶性VCAM(Rose et al. (2000) Blood 95:602-609)；可溶性VCAM断片(Dudgeon et al., Eur. J. Biochem. (1994) 226(2):517-23)；VCAMペプチド配列RTQIDSPLN(SEQ ID NO:44)、TQIDSP(SEQ ID NO:45)、QIDS(SEQ ID NO:46)、IDSP(SEQ ID NO:47)およびKLEK(SEQ ID NO:48)(Clements et al., J. Cell Sci. (1994) 107(Pt 8):2127-35)により例示されるヘビのディスインテグリンを含む。 Exemplary peptides that inhibit the specific binding of integrin α4β1 to one or more of its ligands include, but are not limited to:

(X is any amino acid or modified amino acid), (X) C ^* (X) PC ^* (SEQ ID NO: 31) (X is any amino acid), RC ^* DPC ^* (SEQ ID NO: 32 ), C ^* WLDVC ^* (SEQ ID NO: 33), YC ^* APC ^* (SEQ ID NO: 34) and YC ^* DPC ^* (SEQ ID NO: 35), and phenylacyl-C ^* DfC ^* (SEQ ID NO : 36) (“f” is D-Phe) (Jackson et al., J. Med. Chem. (1997) 40 (21): 3359-68); RC ^* D (ThioP) C ^* (SEQ ID NO: 37) (Nowlin et al., J. Biol. Chem. (1993) Sep 25, 268 (27): 20352-9); 9-fluorene carboxyl RC ^* D (ThioP) C ^* (SEQ ID NO: 38 ) (Cardarelli et al., J. Biol. Chem. (1994) 269 (28): 18668-73); EGYYGNYGVYA (SEQ ID NO: 39) and C ^* YYGNC ^* (SEQ ID NO: 97) ( ^* is a ring) As well as their modifications (Thorsett et al., Inhibitors of leukocyte adhesion (1996) WO9602644); 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (SEQ ID NO: 41) (Cardarelli et al., J. Biol. Chem. (1994) 269 (28): 18668-73). Other exemplary peptides include, but are not limited to, EC3 from carpet viper (Echis carinatus), a subunit of EC3, and the sequence

EC3B, a peptide fragment of EC3, and modifications thereof (Brando et al., Biochem. Biophys. Res. Commun. (2000) 267 (1): 413-417, and Marcinkiewicz et al., J. Biol. Chem. (1999) 274 (18): 1 2468-73); soluble VCAM (Rose et al. (2000) Blood 95: 602-609); soluble VCAM fragment (Dudgeon et al., Eur. J. Biochem. 1994) 226 (2): 517-23); VCAM peptide sequences RTQIDSPLN (SEQ ID NO: 44), TQIDSP (SEQ ID NO: 45), QIDS (SEQ ID NO: 46), IDSP (SEQ ID NO: 47) And snake disintegrin, exemplified by KLEK (SEQ ID NO: 48) (Clements et al., J. Cell Sci. (1994) 107 (Pt 8): 2127-35).

  Further exemplary modified peptides that inhibit the specific binding of integrin α4β1 to one or more of its ligands are peptidomimetics (ie, organic molecules that mimic the structure of the peptide); or peptoids such as vinylic peptoids including. Examples of cyclic peptides and peptidomimetics that are within the scope of the present invention include, but are not limited to, the peptide structure GPEYLDVP (SEQ ID NO: 49), such as a compound named TBC722 (Kogan et al., WO9600581). Peptide structure ILDV (SEQ ID NO: 51), based on peptide structure LDVP (SEQ ID NO: 50), including phenylacetyl LDFp (Arrhenius et al., WO9515973; Arrhenius et al., WO9606108) Dutta, WO9702289), BIO1211 (4- (2-methylphenyllulyed) phenylacetyl LDVP) BIO1272 (Lin et al., WO9200995; Lin et al., WO9622966), CY9652 a CS-1 peptide mimic, TBC3342, ZD -7349 (Curley et al., (1999) Cell. Mol. Life Sci., 56: 427-441); and others (EP-842943-A2, WO9842656-A1, WO9620216-A1, WO9600581-A1, Souers et al. (1998) Bioorg. Med. Chem. Lett., 8: 2297-2302). Exemplary peptides and modified peptides are shown in FIG. 5 (see Lin et al. (1999) J. Med. Chem., 42: 920-934), FIG. 6 (Lin et al. (1998) Curr. Opin. Chem. Biol., 2: 453-457) and FIG. 7 (see Souers et al. (1998) Bioorg. Med. Chem. Lett., 8: 2297-2302). Methods for generating libraries of mimetics and methods for evaluating libraries of mimetics for inhibiting the binding of receptors to their ligands are known in the art. (Souers et al. (1998) supra).

  Other peptides useful as α4β1 antagonists that reduce angiogenesis can be identified by screening a library of peptides that can be purchased from commercial sources and prepared using well-known methods of chemical synthesis ( Koivunen et al. J. Cell Biol., 124: 373-380 (1994)). For example, peptide agonists of integrin α4β1 other than those specifically disclosed herein are as described in US Pat. No. 5,780,426 to Palladino et al., The entire contents of which are hereby incorporated by reference. Can be identified using methods known in the art, such as by panning a phage display peptide library. For example, a phage display peptide library is panned with an integrin α4β1 receptor attached to a solid support such as a small diameter (1 μm) polystyrene latex bead. Phage selected by this method can then be tested for specific binding to integrin α4β1 by ELISA or other immunologically based assays. Individual peptide sequences are then determined by sequencing of phage DNA. Further analysis of the minimal peptide sequence required for binding can be assessed by deletion and site-directed mutagenesis followed by phage testing for binding to integrin α4β1 by ELISA. As the identified peptide candidates are fused to the major phage coat protein, soluble peptides are then chemically synthesized and the activity of these free peptides varies in vitro and in their ability to act as antagonists of the integrin α4β1 receptor. Tested in an in vivo assay.

3. Nucleic Acid Sequence In an alternative embodiment, the agent that inhibits specific binding of α4β1 to one or more of its ligands is a nucleic acid sequence. The terms “nucleic acid sequence” and “nucleotide sequence” as used herein refer to two or more nucleotides covalently linked to each other. Included within this definition are oligonucleotides, polynucleotides, and fragments and / or portions thereof, genomic and / or synthetic, which can be single stranded or double stranded, and which represents the sense or antisense strand DNA and / or RNA of origin. Nucleic acid sequences that are particularly useful in the present invention include, but are not limited to, antisense sequences and ribozymes. The nucleic acid sequence is intended to bind to the genomic DNA or RNA sequence encoding integrin α4β1 or one or more of its ligands, thereby inhibiting the binding of integrin α4β1 to one or more of its ligands. The Antisense and ribozyme sequences can be delivered to cells by transfecting the cells with a vector that expresses the ribozyme as an antisense nucleic acid or mRNA molecule. Alternatively, delivery can be achieved by capturing ribozyme and antisense sequences in liposomes.

Antisense sequences Antisense sequences have been found in several genes (Markus-Sekura (1988), including the gene encoding VCAM1, one of the integrin α4β1 ligands (US Pat. No. 6,252,043, incorporated by reference in its entirety). ) Anal. Biochem. 172: 289-295; Hambor et al. (1988) J. Exp. Med. 168: 1237-1245; and patent EP 140 308). The terms “antisense DNA sequence” and “antisense sequence” when used interchangeably herein refer to the sequence of deoxyribonucleotide residues in relation to the sequence of deoxyribonucleotide residues in the sense strand of a DNA duplex. Refers to the deoxyribonucleotide sequence in the reverse 5 'to 3' orientation. The “sense strand” of a DNA duplex refers to the strand in the DNA duplex that is transcribed into “sense mRNA” by the cell in nature. The sense mRNA is generally finally translated into a polypeptide. Thus, an “antisense DNA sequence” has the same sequence as the non-coding strand in a DNA duplex and is an “antisense RNA” (ie, a ribonucleotide sequence whose sequence is complementary to the “sense mRNA” sequence). Is an array encoding. The symbol (−) (ie, “minus”) is sometimes used for the antisense strand, and the symbol (+) is sometimes used for the sense (ie, “plus”) strand. Antisense RNA can be made by any method, including synthesis by joining an antisense DNA sequence to a promoter that allows synthesis of the antisense RNA. The transcribed antisense RNA strand binds to the natural mRNA produced by the cell to form a duplex. These duplexes then block either further transcription of the mRNA or its translation or facilitate its degradation.

  Any antisense sequence is either (a) not treated with an antisense integrin α4β1 or integrin α4β1 ligand sequence, (b) treated with a sense integrin α4β1 or integrin α4β1 or integrin α4β1 ligand sequence, or ( c) reducing one or more levels of integrin α4β1 and / or its ligands (eg, VCAM and fibronectin) to an amount that is less than the amount of integrin α4β1 or integrin α4β1 ligand expression in control tissues treated with nonsense sequences. Can be considered to be within the scope of the present invention.

  The terms “reducing the level of integrin α4β1 or integrin α4β1 ligand expression”, “decrease integrin α4β1 or integrin α4β1 ligand expression” and their grammatical equivalents preferably refer to the level of integrin α4β1 or integrin α4β1 ligand expression, preferably Is 20% less than the amount in the control tissue, more preferably 50% less than the amount in the control tissue, even more preferably 90% less than the amount in the control tissue, and most preferably Western blot analysis of integrin α4β1 or integrin α4β1 ligand , Immunofluorescence for detection of integrin α4β1 or integrin α4β1 ligand, reverse transcription polymerase chain reaction (RT-PCR) for detection of integrin α4β1 or integrin α4β1 ligand mRNA, or integrin In situ hybridization for detection of 4β1 or integrin α4β1 ligand mRNA, a background level, or some points to be reduced to an amount that can not be detected. If background or undetectable levels of integrin α4β1 or integrin α4β1 ligand peptide or mRNA are measured, this may indicate that integrin α4β1 or integrin α4β1 ligand is not expressed. A decrease in the level of integrin α4β1 or integrin α4β1 ligand may mean, but is not required, the absolute absence of expression of integrin α4β1 or integrin α4β1 ligand. The present invention does not require, and is not limited to, an antisense integrin α4β1 or integrin α4β1 ligand sequence that eliminates integrin α4β1 or integrin α4β1 ligand expression.

  An antisense integrin α4β1 or integrin α4β1 ligand sequence capable of reducing the level of integrin α4β1 expression is at least one of the integrin α4β1 cDNA or the integrin α4β1 ligand cDNA under, for example, high stringency or moderate stringency conditions. A sequence capable of hybridizing to a portion. Antisense integrin α4β1 sequences and antisense integrin α4β1 ligand sequences within the scope of the present invention can be designed using approaches known in the art. In a preferred embodiment, the antisense integrin α4β1 and antisense integrin α4β1 ligand sequences are designed to hybridize to the integrin α4β1 mRNA or integrin α4β1 ligand mRNA encoded by the coding regions of the integrin α4β1 gene and the integrin α4β1 ligand gene, respectively. Is done. Alternatively, antisense integrin α4β1 or integrin α4β1 ligand sequences can be designed to reduce transcription by hybridizing to upstream untranslated sequences, thereby preventing promoter binding to transcription factors.

  In preferred embodiments, the antisense oligonucleotide sequences of the invention range in size from about 8 nucleotide residues to about 100 nucleotide residues. In an even more preferred embodiment, the oligonucleotide sequence ranges in size from about 8 nucleotide residues to about 30 nucleotide residues. In the most preferred embodiment, the antisense sequence has 20 nucleotide residues.

  However, the present invention is not intended to be limited to the number of nucleotide residues in the oligonucleotide sequences disclosed herein. Any oligonucleotide sequence capable of reducing the expression of integrin α4β1 or integrin α4β1 ligand is contemplated to be within the scope of the present invention. For example, oligonucleotide sequences can range in size from about 3 nucleotide residues to the entire integrin α4β1 or integrin α4β1 ligand cDNA sequence. One skilled in the art recognizes that the degree of sequence identity decreases with decreasing length, thereby reducing the specificity of the oligonucleotide for integrin α4β1 mRNA or integrin α4β1 ligand mRNA.

  Antisense oligonucleotide sequences that are useful in the methods of the invention can include naturally occurring nucleotide residues and nucleotide analogs. Nucleotide analogs include, for example, altered sugar moieties, altered intersugar linkages (e.g., phosphodiester linkages of oligonucleotides, sulfur-containing linkages, phosphorothionate linkages, alkyl phosphorothioate linkages, N-alkyl phosphoramidates). Date, phosphorodithionate, alkylphosphonate and substitution with short chain alkyl or cycloalkyl structures), or nucleotide residues containing altered base units. Oligonucleotide analogs are desirable to increase the stability of antisense oligonucleotide compositions under biological conditions, for example, because natural phosphodiester linkages are not resistant to nuclease hydrolysis. Oligonucleotide analogs also incorporate oligonucleotides into liposomes to reduce the amount of antisense oligonucleotides required for therapeutic effect and thereby also reduce the cost of treatment and possible side effects. It may be desirable to improve efficiency and enhance the ability of the composition to penetrate the cell in which the nucleic acid sequence whose activity is to be modulated is located.

  Antisense oligonucleotide sequences can be synthesized using any of a number of methods known in the art and using commercially available services (eg, Genta, Inc.). Synthesis of antisense oligonucleotides can be performed, for example, using a solid support and a commercially available DNA synthesizer. Alternatively, antisense oligonucleotides can also be synthesized using standard phosphoramidate chemistry techniques. For example, for the creation of a phosphodiester bond, oxidation is mediated through iodine, whereas for the synthesis of phosphorothioate, the oxidation is performed using 3H-1,2-benzoate in acetonitrile as a stepwise thiolation of the phosphite bond. Dithiol-3-one, mediated by 1-dioxide. The thiolation step is followed by a capping step, cleavage from the solid support, and purification in HPLC, eg, a gradient of acetonitrile in a PRP-1 column and triethylammonium acetate, pH 7.0.

を含む配列(図1参照)により例示される。 In one embodiment, the antisense DNA sequence is an “integrin α4β1 antisense DNA sequence” (ie, an antisense DNA sequence designed to bind to at least a portion of the integrin α4β1 genomic sequence or to the integrin α4β1 mRNA). is there. The design of the integrin α4β1 antisense DNA sequence is facilitated by the availability of the sequences of the integrin α4 subunit cDNA (FIGS. 8 and 9) and the integrin β1 cDNA (FIG. 10). Particularly preferred antisense sequences are those that hybridize to genomic DNA encoding the portion of integrin α4β1 involved in specific binding to one or more of the ligands, or to RNA. Such integrin α4β1 moieties include, but are not limited to, sequence

Is exemplified by a sequence comprising (see FIG. 1).

を含む。 In another embodiment, the antisense DNA sequence is a “vascular cell adhesion molecule antisense DNA sequence”, ie, an antisense DNA sequence that is designed to bind to at least a portion of a VCAM genomic sequence or to VCAM mRNA. It is. Selection and design of these antisense sequences is made possible by the availability of VCAM cDNA sequences (FIG. 11). Exemplary preferred antisense sequences are those that hybridize to genomic DNA encoding the portion of VCAM (Figure 3A, GenBank accession number P19320) involved in specific binding of VCAM to integrin α4β1, or to RNA It is. At least some examples of VCAM include the amino acid sequence RTQIDSPLNG (SEQ ID NO: 15) (amino acid 60 to amino acid 69); RTQIDSPLSG (SEQ ID NO: 16) (amino acid 348 to amino acid 357), KLEK (SEQ ID NO: 17) (amino acid 103 to amino acid 106, and amino acid 391 to amino acid 394),

including.

、アミノ酸配列LDV(SEQ ID NO:19)(アミノ酸2011位からアミノ酸2013位まで)を含むCS-1配列、アミノ酸配列

を含むCS-5配列をコードするものである。 In yet another embodiment, the antisense DNA sequence is a “fibronectin α4β1 antisense DNA sequence” (ie, an antisense DNA sequence designed to bind to at least a portion of a fibronectin genomic sequence or to fibronectin α4β1 mRNA. ). Selection and design of these antisense sequences is made possible by the availability of sequences for fibronectin cDNA (FIG. 12). An exemplary nucleic acid sequence that can be targeted is the following sequence shown in FIG. 4, the IIICS sequence from amino acid 1982 to amino acid 2111:

, CS-1 sequence including amino acid sequence LDV (SEQ ID NO: 19) (from amino acid 2011 to amino acid 2013), amino acid sequence

Which encodes a CS-5 sequence containing

b. Ribozymes In some alternative embodiments, the agent that inhibits the specific binding of integrin α4β1 to its ligand is a ribozyme. Ribozyme sequences have been successfully used to inhibit the expression of several genes, including the gene encoding VCAM1, one of the integrin α4β1 ligands (US Pat. No. 6,252,043, incorporated in its entirety by reference).

  The term “ribozyme” refers to an RNA sequence that hybridizes to a complementary sequence in a substrate RNA and cleaves the substrate RNA in a sequence-specific manner at the substrate cleavage site. Typically, a ribozyme comprises a “catalytic region” adjacent to two “binding regions”. The ribozyme binding region hybridizes to the substrate RNA, while the catalytic region cleaves the substrate RNA at a “substrate cleavage site”, resulting in a “cleaved RNA product”. The nucleotide sequence of the ribozyme binding region can be completely complementary or partially complementary to the substrate RNA sequence to which the ribozyme binding region hybridizes. Perfect complementarity is preferred to increase specificity and turnover rate (ie, the rate of ribozyme release from the cleaved RNA product). Partial complementarity is less preferred but can be used to design a ribozyme binding region comprising more than about 10 nucleotides. Although intended to be within the scope of the claimed invention, partial complementarity generally has a binding region that has partial complementarity to the substrate RNA that is complete to the substrate RNA. Compared to the specificity and turnover rate of a ribozyme containing a binding region having complementarity, it shows a decrease in the specificity and turnover rate of the ribozyme, which is less preferable than perfect complementarity. Ribozymes can hybridize to partially or completely complementary DNA sequences, but ribozyme cleavage requires 2'-OH on the target molecule not available on the DNA sequence, thus cleaving the hybridized DNA sequence Can not do it.

  The ability of a ribozyme to cleave at a substrate cleavage site can be readily measured using methods known in the art. These methods include, but are not limited to, mutations in a control (e.g., in the absence of a ribozyme or one or both unpaired nucleotide sequences that render the ribozyme incapable of cleaving the substrate RNA. Detection of reduced in vitro or in vivo levels of RNA containing a ribozyme substrate cleavage site for which the ribozyme is specific (e.g., as described herein) as compared to the level of RNA in the presence of a ribozyme sequence comprising Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization, etc.).

  Ribozymes contemplated to be within the scope of the present invention include, but are not limited to, hammerhead ribozymes (eg, Reddy et al., US Pat. No. 5,246,921; Taira et al., US Pat. No. 5,500,357). Goldberg et al., U.S. Pat.No. 5,225,347, the contents of each of which are incorporated herein by reference), Group I intron ribozymes (Kruger et al. (1982) Cell 31: 147-157), ribonuclease P ( Guerrier-Takada et al. (1983) Cell 35: 849-857), hairpin ribozymes (Hampel et al., US Pat. No. 5,527,895), and hepatitis delta virus ribozyme (Wu et al. (1989) Science 243: 652-655).

  Ribozymes can be designed to cleave at any substrate RNA at the substrate cleavage site, as long as the substrate RNA contains one or more substrate cleavage sequences and the sequence adjacent to the substrate cleavage site is known. In effect, the in vivo expression of such ribozymes, and the resulting cleavage of the RNA transcript of the gene of interest, reduces or eliminates the expression of the corresponding gene.

  For example, if the ribozyme is a hammerhead ribozyme, the basic principles of hammerhead ribozyme design are selection of a region in the substrate RNA that contains the substrate cleavage sequence, two antisense oligonucleotides that hybridize to sequences adjacent to the substrate cleavage sequence. Creating one sequence (ie, a binding region) and placing a sequence that forms a hammerhead catalytic region between the two binding regions.

  In order to select a region in the substrate RNA that contains the candidate substrate cleavage site, the sequence of the substrate RNA needs to be determined. The sequence of RNA encoded by the genomic sequence of interest can be readily determined using methods known in the art. For example, RNA transcript sequences can be used manually or using available computer programs (e.g. GENEWORKS from IntelliGenetic Inc. or RNADRAW available from the Internet at ole@mango.mef.ki.se). Either can be achieved by changing T in the DNA sequence encoding the RNA transcript to U.

  The substrate cleavage sequence in the target RNA can be located by searching the RNA sequence using available computer programs. For example, if the ribozyme is a hammerhead ribozyme, the catalytic region of the hammerhead ribozyme is NUH (N is any nucleotide, U is uridine, H is cytosine (C), uridine (U) or adenine (A ) But not guanine (G)) is known in the art. The U-H doublet at the NUH cleavage site does not include the U-G doublet because G pairs with the adjacent C in the ribozyme and prevents ribozyme cleavage. Typically N is G and H is C. As a result, GUC was found to be the most efficient substrate cleavage site for hammerhead ribozymes, but ribozyme cleavage in CUC is also efficient.

  In preferred embodiments, the substrate cleavage sequence is located in the loop structure of the substrate RNA or in an unpaired region. Computer programs for the prediction of RNA secondary structure formation are known in the art, such as “RNADRAW”, “RNAFOLD” (Hofacker et al. (1994) Monatshefte F. Chemie 125: 167-188; McCaskill (1990) Biopolymers 29: 1105-1119), “DNASIS” (Hitachi) and “THE VIENNA PACKAGE”.

  In addition to the desirability of selecting a substrate cleavage sequence located in the loop structure or unpaired region of the substrate RNA, the substrate cleavage sequence prevents the translated truncated polypeptide from being biologically functional. In addition, it is not necessary, but desirable, to be located downstream (ie, at the 3 ′ end) of the translation initiation codon (AUG or GUG).

を含む配列(図1参照)により例示される。 In a preferred embodiment, the ribozyme is an “integrin α4β1 ribozyme” (i.e., the substrate cleavage sequence is designed to hybridize with a portion of integrin α4β1 that is involved in the specific binding of integrin α4β1 to one or more of its ligands. Ribozyme). Such integrin α4β1 moieties include, but are not limited to, sequence

Is exemplified by a sequence comprising (see FIG. 1).

  In an alternative embodiment, the substrate cleavage sequence is designed to hybridize with a portion of the integrin α4β1 ligand, which portion is responsible for specific binding of the ligand to the integrin α4β1.

を含む。 In a more preferred embodiment, the ribozyme is a “vascular cell adhesion molecule ribozyme” (ie, a ribozyme whose substrate cleavage sequence is designed to hybridize to the portion of VCAM involved in specific binding of VCAM to integrin α4β1). It is. An exemplary portion of the ligand VCAM (FIG. 3A, GenBank Accession No. P19320) is the amino acid sequence RTQIDSPLNG (SEQ ID NO: 15) (amino acid 60 to amino acid 69); RTQIDSPLSG (SEQ ID NO: 16) (amino acid 348 to amino acid 357), KLEK (SEQ ID NO: 17) (amino acid 103 to amino acid 106, and amino acid 391 to amino acid 394),

including.

、アミノ酸配列LDV(SEQ ID NO:19)(アミノ酸2011位からアミノ酸2013位まで)を含むCS-1配列、アミノ酸配列REDV(SEQ ID NO:20)(アミノ酸2091位からアミノ酸2093位まで)を含むCS-5配列、IDAPS(SEQ ID NO:21)(アミノ酸1903位からアミノ酸1907位まで)、

を含む。 In an alternative preferred embodiment, the ribozyme is a “fibronectin ribozyme” (ie, a ribozyme whose substrate cleavage sequence is designed to hybridize to a portion of fibronectin involved in specific binding of fibronectin to integrin α4β1). . An exemplary ligand fibronectin moiety includes the following sequence shown in FIG. 4, the IIICS sequence from amino acid 1982 to amino acid 2111:

A CS-1 sequence containing the amino acid sequence LDV (SEQ ID NO: 19) (from amino acid 2011 to amino acid 2013), including the amino acid sequence REDEV (SEQ ID NO: 20) (from amino acid 2091 to amino acid 2093) CS-5 sequence, IDAPS (SEQ ID NO: 21) (from amino acid position 1903 to amino acid position 1907),

including.

  It is known in the art that the specificity of ribozyme cleavage for substrate RNA molecules is determined by the sequence of nucleotides adjacent to the substrate cleavage site and hybridizing to the ribozyme binding region. Thus, the ribozyme will cleave at different positions within the substrate RNA molecule by altering the sequence of the binding region surrounding the ribozyme catalytic region of the ribozyme so that the binding region hybridizes to any known sequence on the substrate RNA. Can be designed.

  In addition to changing the sequence of the binding region to result in binding to different positions on the RNA substrate, the number of nucleotides in each of the ribozyme binding regions also alters the specificity of the ribozyme for a given position on the RNA substrate. Can be changed for. The number of nucleotides in the binding region is preferably between about 5 and about 25 nucleotides, more preferably between about 11 and about 15 nucleotides, even more preferably between about 7 and about 10 nucleotides.

  Those skilled in the art recognize that the two binding regions adjacent to the ribozyme catalytic region need not be of equal length. A binding region comprising any number of nucleotides is contemplated to be within the scope of the present invention so long as the desired specificity of the ribozyme for the RNA substrate and the desired cleavage rate of the RNA substrate are achieved. One skilled in the art will recognize that longer nucleotide sequence binding regions increase specificity for a particular substrate RNA sequence, but after cleavage, dissociate from the substrate RNA and reduce the ability of the ribozyme to bind to another substrate RNA molecule. And the cutting rate can be reduced accordingly. On the other hand, binding regions with shorter nucleotide sequences can have a higher rate of dissociation and cleavage, but specificity for the substrate cleavage site is impaired.

  It is well within the skill in the art to determine the optimal length for the ribozyme binding region so that the desired specificity and cleavage rate is achieved. Both the specificity of the ribozyme for substrate RNA and the rate of cleavage of the substrate RNA by the ribozyme can be measured, for example, by kinetic studies combined with Northern blot analysis or nuclease protection assays.

  In preferred embodiments, the complementarity between the ribozyme binding region and the substrate RNA is perfect. However, the present invention is not limited to ribozyme sequences whose binding region exhibits complete complementarity with substrate RNA. The complementarity may be partial as long as the desired specificity of the ribozyme for the substrate cleavage site and the rate of cleavage of the substrate RNA are achieved. Thus, as long as substantial base pairing with substrate RNA is maintained in the region adjacent to the substrate cleavage sequence and base pairing with the substrate cleavage sequence is minimal, base changes can occur in one or both ribozyme binding regions. Can be made in The term “substantial base pairing” refers to more than about 65%, more preferably more than about 75%, and even more preferably more than about 90% of the bases of a hybridized sequence is base pairing. Means that.

  It may be desirable to increase the intracellular stability of a ribozyme expressed by an expression vector. This is accomplished by designing a ribozyme that is expressed to include a secondary structure (eg, a stem-loop structure) within the ribozyme molecule. Secondary structures suitable for stabilizing ribozymes include, but are not limited to, stem-loop structures formed by intrastrand base pairs. An alternative to using a stem-loop structure to protect the ribozyme against ribonuclease degradation is the insertion of a stem loop at each end of the ribozyme sequence (Sioud and Drlica (1991) Proc. Natl. Acad. Sci. USA 88 : 7303-7307). Other secondary structures that are useful in reducing the sensitivity of ribozymes to ribonuclease degradation are hairpins, bulge loops, inner loops, multi-branched loops, and "Molecular and Cellular Biology," Stephen L. Wolfe (Ed.), Wadsworth Includes pseudonodule structures as described in Publishing Company (1993) p. In addition, cyclization of the ribozyme molecule protects against ribonuclease degradation since exonuclease degradation is initiated at either the 5 ′ or 3 ′ end of the RNA. Methods for expressing circular RNA are known in the art (see, eg, Puttaraju et al. (1993) Nucl. Acids Res. 21: 4253-4258).

  Once a ribozyme having the desired binding region, catalytic region, and nuclease stability has been designed, the ribozyme can be made by any known means, including chemical synthesis. The chemically synthesized ribozyme can be introduced into cells by, for example, microinjection, electroporation, lipofection and the like. In a preferred embodiment, the ribozyme is produced by expression from an expression vector containing a gene encoding the desired ribozyme sequence.

4. Other Agents While the present invention is exemplified herein using antibodies, peptides and nucleic acid sequences that inhibit the specific binding of integrin α4β1 to one or more of its ligands, Clearly, other agents (e.g., organic molecules, inorganic molecules, etc.) are within the scope, as long as the agent can inhibit the specific binding of integrin α4β1 to one or more of its ligands. Is contemplated. Such agents can be identified by screening a library of test compounds (made as described below) using competitive binding assays or cell adhesion assays. In competitive binding assays, for example, integrin α4β1 is coated on a plastic microtiter plate and contacted with a labeled known integrin α4β1 ligand (eg, CS-1 fibronectin or VCAM). Test compounds are tested for their ability to inhibit the binding of labeled ligand to integrin α4β1. Compounds that inhibit such binding are identified as agents capable of inhibiting the specific binding of integrin α4β1 to the ligand.

  Alternatively, in a cell adhesion assay, a labeled known integrin α4β1 ligand (e.g., CS-1 fibronectin or VCAM) is coated on a culture plate and cells expressing integrin α4β1 are allowed to migrate in the presence of a library of test compounds. Let it adhere to the ligand for 20-30 minutes. Compounds that inhibit the binding of integrin α4β1 expressing cells to the coating of integrin α4β1 ligand are identified as agents that inhibit the specific binding of integrin α4β1 to the ligand.

C. Integrin α4β1 mediates the transport of endothelial progenitor cells, as exemplified by endothelial stem cells, during neovascularization Bone marrow-derived stem cells, including vascular endothelium, smooth muscle, neurons and muscles, regenerate tissues that undergo repair. Contributes to proliferation (Asahara et al., Science 1997 Feb 14; 275 (5302): 964-7; Jain et al., Cancer Cell 2003 Jun; 3 (6): 515-6; Religa et al., Transplantation 2002 Nov 15; 74 (9): 1310-5; Priller et al., J Cell Biol. 2001 Nov 26; 155 (5): 733-8; LaBarge et al., Cell 2002 Nov 15; 111 (4): 589 -601). The mechanism by which hematopoietic stem cells home to the site of ongoing tissue repair remains unclear. Here, the inventors have shown that integrin α4β1 (VLA-4) promotes the migration of endothelial progenitor cells (EPCs) from the circulation to the site of angiogenesis. During angiogenesis, integrin α4β1 promotes adhesion of EPC to VCAM in activated endothelium and to alternatively spliced tissue (CS-1) fibronectin found to be under this endothelium . Antagonists of α4β1 block EPC efflux from the circulation during angiogenesis, thereby inhibiting growth factors and tumor-induced angiogenesis in vivo. Thus, α4β1 contributes to angiogenesis by controlling the recruitment of hematopoietic stem cells to the new vascular bed.

  Neovascularization is an important component of the tissue repair process that contributes to wound healing, but when chronically stimulated it also plays a role in pathologies such as tumor growth and inflammatory diseases (Carmeliet et al. al., Nat Med. 2003 Jun; 9 (6): 653-60; Carmeliet et al., Nature 2000 Sep 14; 407 (6801): 249-57). Neovascularization is thought to occur by two mechanisms. Activation of quiescent endothelial cells in tissue vessels by angiogenic growth factors promotes the development of new blood vessels by budding (Carmeliet et al., Nat. Med. 2003 Jun; 9 (6): 653-60; Carmeliet et al., Nature 2000 Sep 14; 407 (6801): 249-57). The second mechanism involves the homing of bone marrow-derived endothelial stem cells to sites of neovascularization such as ischemic limbs or tumors (Asahara et al., Science 1997 Feb supra; Jain et al., Cancer Cell 2003 Jun; 3 (6): 515-6; Lyden et al., Nat. Med. 2001 Nov; 7 (11): 1194-201; Takahashi et al., Nat. Med. 1999 Apr; 5 (4): 434-8 ; Kawamoto et al., Circulation 2001 Feb 6; 103 (5) 634-7; Hattori et al., J Exp Med 2001 May 7; 193 (9): 1005-14; Kalka et al., Proc Natl Acad Sci USA 2000 Mar 28; 97 (7): 3422-7). These bone marrow-derived stem cells can home to muscle, brain, and other tissues undergoing repair that are involved in tissue regeneration (Asahara et al., Science 1997 Feb supra; Jain et al., Cancer Cell 2003 Jun; 3 (6) 515-6; Religa et al., Transplantation 2002 Nov 15; 74 (9): 1310-5; Priller et al., J Cell Biol 2001 Nov 26; 155 (5): 733- 8; LaBarge et al., Cell 2002 Nov 15; 111 (4): 589-601; Lyden et al., Nat Med 2001 Nov; 7 (11): 1194-201; Takahashi et al., Nat Med 1999 Apr; 5 (4): 434-8; Kawamoto et al., Circulation 2001 Feb 6; 103 (5): 634-7; Hattori et al., J Exp Med 2001 May 7; 193 (9): 1005-14; Kalka et al., Proc Natl Acad Sci USA 2000 Mar 28; 97 (7): 3422-7; Torrente et al., J Cell Biol 2003 Aug 4; 162 (3): 511-20). However, the mechanism by which bone marrow-derived stem cells such as EPC exit the circulation and enter the tissue to participate in the tissue repair process remains unclear.

  Antagonists of integrin α4β1 (antibodies, peptides, etc.) are found in the extracellular matrix in bone marrow derived stem cells or progenitor cells by entering their tissues, blocking their association with the vascular endothelium, and in tissues below the endothelium. Inhibiting them by blocking their migration. These antagonists can be used to block hematopoietic stem cells from participating in angiogenesis, atherosclerosis, restenosis, inflammation, cancer, and other diseases in which hematopoietic stem cells play a role. In addition, reagents that selectively bind to α4β1, such as high-affinity antibodies, recombinant soluble VCAM or CS-1 fibronectin, can cause hematopoietic stem cells, they can be proliferated, repair damaged heart tissue, ischemic tissue It can be used to purify from tissue, bone marrow, peripheral blood, umbilical cord blood, etc., so that it can be further used for therapeutic applications such as stimulation of angiogenesis and repair of congenital muscle defects. Finally, cytokines that upregulate VCAM in the vascular endothelium can be used to promote the influx of hematopoietic stem cells into the tissue by providing a site for hematopoietic stem cells to adhere to the vascular endothelium.

  Currently, bone marrow derived hematopoietic stem cells are under investigation for use in muscle, heart, ischemic tissue, nerve repair, and myriad other creative applications. Researchers can show that purified or native bone marrow-derived hematopoietic stem cells enter normal tissue, but generally, the number of cells that reach it to the tissue is small. In addition, bone marrow derived hematopoietic stem cells are involved in pathological processes such as tumor growth and angiogenesis, atherosclerosis and restenosis. The data herein (as in Examples 4-15) shows the identification of the molecular pathways by which these cells recognize the endothelium, adhere to it and enter the tissue. Furthermore, the present inventors have determined several methods for inhibiting or promoting hematopoietic stem cell homing.

  Since the integrin α4β1 is also an effector of immune cell transport in vivo, the data herein (Examples 4-15) show that α4β1 is a putative precursor common to the “hemangioblast”, HSC and EPC strains Suggests that it can be expressed by the body. The data herein shows that integrin α4β1 plays an important and unique role in mediating the tissue repair process by mediating the interaction of endothelial progenitor cells with the more established endothelium. The integrin α4β1 may also play an important role in controlling endothelial sprouting from established blood vessels; its transient expression on new blood vessels may indicate a functional role early in the angiogenic process. Since CD34-positive bone marrow-derived stem cells are integrin α4β1-positive, it is possible for this integrin to control the transport of other CD34-positive stem cells to tissues during tissue repair. These data indicate that integrin α4β1 antagonists can be used to suppress pathological angiogenesis and tumor growth, as well as other pathological conditions in which hematopoietic stem cells play an important role. These data also suggest that homing to tissues in need of hematopoietic stem cell repair can be enhanced by stimulating the endothelium to express VCAM and by stimulating hematopoietic stem cell α4β1 activity To do.

  Little is known about the mechanism by which hematopoietic stem cells exit the circulation and enter the tissue. Furthermore, there are no known methods for blocking or enhancing this process. Thus, the present invention provides the only known method for blocking or promoting homing of hematopoietic stem cells to tissue. The present invention is useful in blocking homing by using inhibitors of integrin α4β1, a hematopoietic stem cell receptor for vascular endothelium. The invention is also useful in stimulating homing by expressing VCAM, a counter-receptor on the endothelium (by administering growth factors or inflammatory cytokines to regional vasculature). is there.

  Integrin α4β1 antibodies, peptides or inhibitors of organic molecules can cause hematopoietic stem cells to enter tissues in vivo and to prevent pathological angiogenesis in cancer, arthritis and neovascular eye diseases, atherosclerosis, restenosis, etc. Can be used to inhibit involvement in such abnormal tissue repair processes. Stimulation of VCAM expression in the endothelium of tissues in need of repair can be used to promote homing of hematopoietic stem cells to the tissue. Finally, reagents that bind α4β1 with high affinity can be used to purify or isolate hematopoietic stem cells for use in therapeutic applications.

  The data herein (eg, Examples 4-15) show that inhibiting α4β1 blocks angiogenesis (growth factors and tumor inducibility), blocks endothelial stem cell proliferation in vitro, endothelial stem cells in vitro Have been shown to block adhesion to the endothelium and to block recruitment of endothelial stem cells to the endothelium in vivo. The data herein (eg, Examples 4-15) also shows that all bone marrow-derived stem cells (which can be identified by the expression of CD34 positive markers) express α4β1 and block α4β1, indicating that additional stem cells It is currently showing that it adheres to the endothelium and blocks it from entering the tissue. These studies show the potential of isolating stem cells using α4β1 expression in combination with additional stem cell markers.

  The present invention is useful by utilizing the inhibition of integrin α4β1: other neovascular diseases such as suppression of tumor growth, arthritis, eye diseases and psoriasis by blocking hematopoietic stem cell contribution to the disease Suppression of atherosclerosis and restenosis.

  The present invention is also useful by taking advantage of the enhanced influx of hematopoietic stem cells into tissues by inducing the expression of VCAM on the endothelium, a counter-receptor for α4β1: ischemic disease (heart attack, diabetes ) Enhancement of angiogenesis, enhancement of muscle and nerve repair in neuromuscular diseases, enhancement of other types of tissue repair. The present invention is also useful for isolating hematopoietic stem cells using integrin α4β1 selection.

D. Altering Adhesion, Migration and Differentiation of Hematopoietic Progenitor Cells The present invention further provides adhesion and / or HPC to target tissues by using agents that alter the specific binding of integrin α4β1 to its ligand. Methods are provided for altering migration as well as altering differentiation of HPC to secondary cell types. The present invention promotes homing of exemplary circulating hematopoietic stem cells to the integrin α4β1 (VLA-4) expressed on a new vascular structure, α4β1 ligand, vascular cell adhesion molecule (VCAM) and cellular fibronectin It is premised at least in part on the surprising discovery (Examples 17-24). CD34 + stem cells expressing integrin α4β1 homed to sites of active neovascularization, but not to normal tissues. Antagonists of integrin α4β1 blocked exemplary hematopoietic stem cells from attaching to the endothelium in vitro and in vivo, and their growth into new blood vessels (Examples 17-24).

  The term `` cell adhesion '' as used herein is an extracellular matrix (e.g., fibronectin, collagen I-XVIII, laminin, vitronectin, fibrinogen, osteopontin, Del 1, tenascin, von Willebrand factor, etc.) To multiple components, to ligands expressed on the cell surface (e.g. VCAM, ICAM, LI-CAM, VE-cadherin, integrin α2, integrin α3, etc.) and / or of the same type (e.g. , Adhesion of HPC to another HPC) or another type of cells (e.g., HPC, endothelial cells, endothelial stem cells, stem cells expressing CD34, fibroblasts, stromal cells, tumor cells, etc.) Refers to the physical contact of cells.

  The term `` reducing cell adhesion '' means that the level of adhesion is preferably 10% less, more preferably 50% less, even more preferably 75% less, even more preferably 90% than the amount in control cells. Less and most preferably refers to a reduction by an amount that is the same level observed in control cells. Although a decrease in the level of cell adhesion may mean an absolute lack of cell adhesion, it is not necessary. The present invention does not require and is not limited to a method that completely eliminates cell adhesion. The level of cell adhesion can be measured using the methods disclosed herein and others known in the art (eg, Varner WO 03/019136 A3).

  The term `` cell migration '' as used herein refers to one or more components of the extracellular matrix (e.g., fibronectin, collagen I-XVIII, laminin, vitronectin, fibrinogen, osteopontin, Del 1, tenascin, Across other cells of the same type (e.g., migration of HPC along another HPC) and / or of different cells (e.g., endothelial cells, endothelium) HPC migration along stem cells, CD34 expressing stem cells, fibroblasts, stromal cells, tumor cells, etc.), refers to the translocation of cells along the surface. Thus, “transendothelial migration” of a cell refers to the translocation of cells across one or more components of the extracellular matrix and / or cells of endothelial tissue.

  The term “reducing cell migration” means that the level of cell migration is preferably 10% less, more preferably 50% less, even more preferably 75% less, and even more preferred than the amount in control cells. Refers to a 90% less, and most preferably, a reduction in an amount that is the same level observed in control cells. A decrease in the level of cell migration may mean an absolute lack of cell migration, but it is not necessary. The present invention does not require and is not limited to a method that completely eliminates cell migration. Cell migration levels are determined using methods disclosed herein and known in the art such as time-lapse video microscopy, curettage wound assays, etc. (e.g., Varner WO 03/019136 A3). Can be measured.

  `` Level of differentiation '' when referring to cells of interest in a sample is differentiated cells (e.g., B cells expressing the B220 marker, T cells expressing the CD3 marker, and myeloid cells expressing CD11b, respectively) Refers to the amount per cell of interest (eg, hematopoietic progenitor cells) of the expressed differentiation marker (eg, B220, CD3, CD11b, etc.) compared to the amount per cell of the same marker expressed by

  The term “reducing cell differentiation” means that the level of cell differentiation is preferably 10% less, more preferably 50% less, even more preferably 75% less, and even more preferred than the amount in control cells. Refers to a 90% less, and most preferably, a reduction in an amount that is the same level observed in control cells. A decrease in the level of cell differentiation may mean an absolute lack of cell differentiation, but it is not necessary. The present invention does not require and is not limited to a method that completely eliminates cell differentiation. The level of cell differentiation can be measured using methods disclosed herein and known in the art.

E. Altering Hematopoietic Progenitor Cell Adhesion, Migration and Differentiation The present invention relates to a method of altering the binding of α4β1 to one or more of its ligands (eg, fibronectin and VCAM) in a tissue in a subject. Provides a method for altering HPC adhesion, migration and / or differentiation. In one embodiment, the subject has a condition associated with undesirable HPC adhesion, migration and / or differentiation, such as in angiogenic diseases. The term “angiogenic disease” is used broadly herein to mean any condition characterized, at least in part, by neovascularization. In contrast, a “non-angiogenic disease” is a condition that is not associated with neovascularization. Angiogenesis is a normal angiogenic process (e.g., during scar formation or conception during wound healing), as well as, but not limited to, neoplasia, diabetic retinopathy and macular degeneration Ocular tissues such as psoriasis and skin diseases such as hemangiomas, gingivitis, rheumatoid arthritis and osteoarthritis, and pathological conditions exemplified by inflammatory bowel disease (e.g., retina, macular) Or cornea), including angiogenesis associated with pathological conditions such as those occurring in skin, as occurring in psoriasis, in synovial tissue, in bone, in intestinal tissue, or in tumors.

  In another embodiment, the subject has a neoplasm. The terms “neoplasm” and “tumor” refer, in part, to tissue growth characterized by angiogenesis. Neoplasms can be benign and are exemplified by, but not limited to, hemangiomas, gliomas, teratomas, and the like. Neoplasms may be malignant or malignant, such as carcinomas, sarcomas, glioblastomas, astrocytomas, neuroblastomas, retinoblastomas and the like.

  The terms “malignant neoplasm” and “malignant tumor” refer to a neoplasm comprising at least one cancer cell. “Cancer cells” have been described earlier (HC Pitot (1978) “Fundamentals of Oncology,” Marcel Dekker (Ed.), New York pp15-28), early and intermediate stages of multistage neoplastic progression. , Or refers to a cell undergoing an advanced phase. The characteristics of early, intermediate and advanced stages of neoplastic progression have been described using microscopy. Cancer cells in each of the three phases of neoplastic progression generally have aberrant karyotypes, including translocations, inversions, deletions, isochromosomes, single chromosomes, and extrachromosomes. Cells in the early stages of malignant progression are termed “hyperplastic cells” and are characterized by dividing uncontrolled and / or at a faster rate than normal cells of the same cell type in the same tissue. Proliferation may be slow or fast, but continues unabated. The intermediate phase of neoplasm progression is called “dysplastic cells”. A dysplastic cell resembles an immature epithelial cell and is generally spatially disrupted within the tissue, losing its specialized structure and function. During the intermediate phase of neoplastic progression, an increasing percentage of epithelium becomes composed of dysplastic cells. “Hyperplastic” and “dysplastic” cells are referred to as “pre-neoplastic” cells. In the advanced phase of neoplastic progression, dysplastic cells become “neoplastic” cells. Tumor cells are typically invasive to other locations in the body where they cause one or more secondary cancers (i.e., they invade adjacent tissue or from the primary site). Either drop off and circulate in the blood and lymph) (ie "metastasis"). Thus, the term “cancer” is used herein to refer to a malignant neoplasm, which may or may not be metastatic. Malignant neoplasms that can be diagnosed using the method of the present invention include, for example, lung cancer, breast cancer, prostate cancer, cervical cancer, pancreatic cancer, colon cancer, ovarian cancer, gastric cancer, esophageal cancer, oral cancer, tongue cancer, gingival cancer. , Skin cancer (e.g., melanoma, basal cell carcinoma, Kaposi sarcoma, etc.), muscle cancer, heart cancer, liver cancer, bronchial cancer, cartilage cancer, bone cancer, testicular cancer, kidney cancer, endometrial cancer, uterine cancer, Bladder cancer, bone marrow cancer, lymphoma cancer, spleen cancer, thymic cancer, thyroid cancer, brain cancer, neuronal cancer, mesothelioma, gallbladder cancer, eye cancer (e.g., corneal cancer, uveal cancer, choroidal cancer, macular) Cancer, vitreous humor cancer, etc.), joint cancer (eg, synovial cancer), glioblastoma, lymphoma, and leukemia. Malignant neoplasms are further exemplified by sarcomas (such as osteosarcoma and Kaposi sarcoma). The present invention specifically contemplates any malignant neoplasm within its scope, so long as the neoplasm is characterized at least in part by angiogenesis associated with α4β1 expression by newly occurring blood vessels.

  The terms “reducing the severity of a pathological condition”, “reducing the severity of a pathological condition” and “reducing symptoms associated with a pathological condition” are harmful It means that a clinical sign or symptom is reduced, delayed or eliminated compared to the level of morbidity in the absence of treatment with a particular composition or method. The effects of reducing the severity of the pathological condition include, but are not limited to, angiography, ultrasound assessment, fluoroscopy, fiber optic endoscopy, biopsy and histology, relative Blood tests that can be used to measure enzyme levels or circulating antigens or antibodies, imaging tests that can be used to detect a decrease in the growth rate or size of a neoplasm, or blood vessels in the retina of a diabetic patient It can be measured by methods routine to those skilled in the art, including ophthalmic procedures that can be used to identify drop in numbers. Such a clinical test is selected based on the particular pathological condition to be treated. For example, the methods of the invention result in a “reduction of tumor tissue” (eg, tumors) compared to control tumor tissue (eg, the same tumor prior to the method of the invention, or a different tumor in a control subject). A decrease in the size, weight and / or volume of the tissue). A decrease in the severity of the morbidity is also a comment made by the patient to be treated, for example, a patient suffering from arthritis feels less pain or has greater joint mobility. Can be detected on the basis of being larger or being more clearly visible to patients with diabetic retinopathy or macular degeneration due to neovascularization.

  A pathological condition amenable to prophylaxis and / or treatment with the methods of the invention includes any pathological condition whose development or progression in the tissue includes HPC adhesion, migration and / or differentiation. Exemplary pathological conditions are, for example, solid tumor cancer, solid tumor metastasis, hemangiofibroma, skin cancer, post-lens fibroplasia, Kaposi's sarcoma, childhood hemangioma, diabetic retinopathy, neovascular glaucoma, age-related Includes macular degeneration, psoriasis, gingivitis, rheumatoid arthritis, osteoarthritis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, and atherosclerotic plaque.

  Other pathological conditions include those involving damage to tissue. The term “damaged” with respect to a tissue refers to a tissue in which the cellular organization of the tissue has changed compared to the cellular organization in normal tissue. Such damage may result from, for example, accidental cuts or destruction of skin tissue (eg, cuts, deep wounds, lacerations) such as burns, surgical-related cuts, and the like. Damaged tissues include lung, breast, prostate, cervix, pancreas, colon, ovary, stomach, esophageal cancer, oral cancer, tongue cancer, gingiva, muscle and the like. In particular, skin damage associated with the formation of undesirable scar tissue is particularly amenable to the therapeutic approach of the present invention.

  Agents that are useful in altering the binding of the integrin α4β1 to the α4β1 ligand include, for example, oral, intranasal or intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, synovial fluid Can be administered by a variety of routes including intrathecally, intraperitoneally, parenterally, including in a bath, or by passive or accelerated absorption through the skin, for example using skin patches or ionphoresis . Still further, the agent can be administered by injection, intubation, through a suppository, orally or topically, the latter passively, for example by direct application of an ointment or powder containing the agent, or May be active, for example, using a nasal spray or inhaler. The agent can also be administered as a topical spray if desired, in which case one component of the composition is a suitable high pressure gas. The pharmaceutical composition can also be incorporated into liposomes, microspheres or other polymer matrices as needed (Gregoriadis, “Liposome Technology,” Vol. 1, CRC Press, Boca Raton, FL 1984). For example, liposomes composed of phospholipids or other lipids are non-toxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. Liposomes are lipid-containing vesicles with lipid bilayers that can take up chemical agents like other lipid carrier particles. Liposomes can be made of one or more phospholipids, but optionally include other substances such as sterols. Suitable phospholipids include phosphatidylcholine, phosphatidylserine, and many others that are well known in the art. Liposomes can be single lamellae, multiple lamellae, or have an undefined lamella structure. For example, in an individual suffering from metastatic carcinoma, the agent in the pharmaceutical composition can be administered intravenously, orally, or by another method of systemically distributing the agent.

  Agents that inhibit the specific binding of integrin α4β1 to one or more of its ligands can be administered in combination with other therapies. For example, in the case of cancer treatment, the agent may be administered in combination with conventional drug therapy and / or chemotherapy directed against solid tumors and for control of the establishment of metastases. In one embodiment, the agent can be administered during or after chemotherapy. In a more preferred embodiment, the agent is administered after chemotherapy when the tumor tissue is about to respond to a toxic challenge. Tumors attempt to induce angiogenesis to recover by supplying blood and nutrients to the tumor tissue. Such recovery is prevented by administration of an agent that inhibits angiogenesis by inhibiting the specific binding of integrin α4β1 to one or more of its ligands. In an alternative embodiment, the agent can be administered after surgery where the solid tumor has been removed as a prophylaxis against future metastases.

  In one embodiment, the agent is administered in a “therapeutic amount” (ie, an amount that is sufficient to achieve the desired result). In particular, a therapeutic amount is an amount that inhibits the specific binding of α4β1 integrin to its specific ligand in the tissue of the subject and results in a reduction, delay or elimination of undesirable pathological effects in the subject. . One skilled in the art recognizes that a “therapeutically effective” amount will vary depending on the therapeutic agent used, the age, condition, and sex of the subject, and the extent of the disease in the subject. In general, the dose should not be so high as to cause adverse side effects such as hyperviscosity syndrome, pulmonary edema, congestive heart failure and the like. The dose can also be adjusted by the individual physician or veterinarian to reach a desired therapeutic goal.

  The therapeutic amount can be measured using in vitro and in vivo assays known in the art and is generally about 0.0001-100 mg / kg body weight.

  The “subject” to which the agent is administered includes, but is not limited to, humans, and monkeys, rodents, sheep, cows, ruminants, rabbits, pigs, goats, horses, cats, birds, etc. Include any animal capable of generating angiogenesis in a tissue, including non-human animals. Preferred non-human animals are rodent members (eg, mice and rats). Thus, the compounds of the invention can be administered by human health professionals and veterinarians.

F. Detection of hematopoietic progenitor cells expressing integrin α4β1 The present invention further detects HPC expressing integrin α4β1 by using an agent that specifically binds to integrin α4β1 polypeptide and / or integrin α4β1 mRNA. Providing a method for These methods identify the presence of HPCs whose adhesion, migration and differentiation are amenable to regulation using the methods of the present invention, where such HPCs are located in normal tissues or in tissues involved in pathological conditions. Useful to identify whether or not. As such, the present invention further provides a method of diagnosing a pathological condition characterized by the involvement of an HPC expressing the integrin α4β1.

  Integrin α4β1 polypeptide can be detected using Western blot analysis or immunofluorescence. Alternatively, the presence of integrin α4β1 mRNA can be detected using reverse transcription polymerase chain reaction (RT-PCR) or in situ hybridization.

  In one embodiment, the agent used in detecting the presence of integrin α4β1 polypeptide and / or mRNA is, for example, whether the specific binding of the agent is to be detected in vivo, or the agent Can be detected by linking the agent to a selected part based on whether tissue suspected of binding is removed and is to be examined ex vivo (e.g., by biopsy) Can be labeled.

  Useful moieties for labeling agonist antagonists include radionuclides, paramagnets, x-ray attenuating substances, fluorescent, chemiluminescent or luminescent molecules, molecules such as biotin, or specific reagents. It can be a molecule that can be visualized in the reaction, for example a substrate for an enzyme or an epitope for an antibody. The moiety can be linked to the agent using well-known methods, selected based in part on the agent and the chemical nature of the moiety. For example, if the moiety is an amino acid sequence such as a hexahistidine (His6) sequence and the agent is a peptide, the His6 sequence can be synthesized as part of the peptide and the His6 labeled agent can be Can be identified by binding of nickel ion reagents.

Methods for chemically linking the moiety to the agent can also be utilized. For example, a method for conjugating a polysaccharide to a peptide uses α- or ε- to NaIO ₄ activated oligosaccharides using squaric acid diester (1,2-diethoxycyclobutene-3,4-dione) as a conjugating reagent. Binding via an amino group, binding via a peptide linker if the polysaccharide has a reducing end and does not contain a carboxyl group (US Pat. No. 5,342,770), synthetic peptide derived from human heat shock protein hsp65 Exemplified, but not limited to, binding to a carrier (US Pat. No. 5,736,146) and using the method of US Pat. No. 4,639,512. Methods for conjugating proteins to proteins include conjugating with a synthetic peptide carrier derived from human heat shock protein hsp65 (U.S. Pat.No. 5,736,146) and methods used to bind peptides to antibodies (US Pat.No. 5,194,254). No. 4,950,480), the method used to attach peptides to insulin fragments (US Pat. No. 5,442,043), the method of US Pat. No. 4,639,512, and EDAC (1-ethyl-3- (3-dimethylaminopropyl) It includes a method (Drabick et al. (1998) Antimicrob. Agents Chemother. 42: 583-588) of linking a cyclic decapeptide polymyxin B antibiotic to an IgG carrier using carbodiimide) -mediated amide formation. Approaches for coupling nucleic acids to proteins are also known in the art, such as those described in US Pat. Nos. 5,574,142; 6,117,631; 6,110,687, each incorporated by reference in its entirety. Methods for conjugating lipids to peptides include, but are not limited to, reductive amination and the use of ether linkages, including secondary or tertiary amines (US Pat. No. 6,071,532), US Pat. No. 4,639,512. The method used to covalently attach peptides to unilamellar liposomes (Friede et al. (1994) Vaccine 12: 791-797), heterobifunctional reagent N-succinimidyl-S-acetylthioacetate (SATA) to bind human serum albumin to liposomes (Kamps et al. (1996) Biochim. Biophys. Acta 1278: 183-190), antibody using phospholipid-poly (ethylene glycol) -maleimide anchor Methods for conjugating Fab 'fragments to liposomes (Shahinian et al. (1995) Biochim. Biophys. Acta 1239: 157-167) and linking Plasmodium CTL epitopes to palmitic acid via cysteine-serine spacer amino acids. How (Verheul et al (1995) J. Immunol Methods 182:.. 219-226) including, have been described in the art.

  The specifically bound agent can be detected in an individual using in vivo imaging methods such as radionuclide imaging, positron emission tomography, computer axial tomography, X-ray or magnetic resonance imaging, Alternatively, after administration, a sample of tissue is obtained from the individual and the specific binding of the agent in the sample is detected (eg, by immunohistochemical analysis; see Varner WO 03/019136 A3). Can be detected.

  An agent that specifically binds to α4β1 integrin in the sample detects the presence of the moiety either directly by detecting the agent or by detecting the radioactivity emitted by the radionuclide moiety. Can be detected indirectly. The specifically bound agent can also contact it further with a reagent that specifically interacts with the agent or with a moiety linked to the agent to detect the interaction of the reagent with the agent or label. Can be detected indirectly. For example, a moiety can be detected by contacting it with an antibody that specifically binds the moiety, particularly if the moiety is linked to an agent. The moiety can also be a substrate that is contacted by an enzyme that interacts with and alters the moiety, for example, such that its presence can be detected. Such indirect detection systems include the use of enzymes such as alkaline phosphatase, horseradish peroxidase, β-galactosidase, etc., but are well known in the art and commercially available, and certain moieties can be The method for incorporating or linking to a particular mold is similar.

G. Compound Screening The present invention also includes the following steps to alter the level of hematopoietic cell adhesion and / or migration and to alter the differentiation of hematopoietic progenitor cells into secondary cell types Provided is a method for screening a test compound for: i) a first composition comprising integrin α4β1, ii) a second composition comprising one or more integrin α4β1 ligands, iii) providing with the test compound B) contacting the test compound with one or more of the first composition and the second composition under conditions for specific binding of the integrin α4β1 to the integrin α4β1 ligand; And c) the integrin α4β1 with the integrin α4β1 ligand in the presence of the test compound as compared to in the absence of the test compound. Changes in the level of heterologous binding are detected, thereby changing the level of the test compound to alter the level of hematopoietic cell adhesion and / or migration, and differentiation of hematopoietic progenitor cells into secondary cell types The stage identified as changing.

  The tissue can be contacted with the agent in vivo or ex vivo (see, eg, US Pat. No. 5,622,699, incorporated by reference). When the screening method of the invention is performed using an in vitro format, it can be adapted to an automated procedure and accordingly identify α4β1 antagonists that may be able to alter HPC adhesion, migration and / or differentiation To enable high throughput screening assays to examine libraries of molecules.

  Alternatively, the screening assay contacts the isolated HPC with a test compound and detects a change in the level of HPC adhesion, migration and / or differentiation, thereby removing the compound from the level of HPC adhesion, migration and / or differentiation. This is done by identifying as changing.

  Test compounds can be generated by methods known in the art for preparing libraries of molecules, such as oligonucleotide libraries (Gold et al., US Pat. No. 5,270,163, incorporated by reference); peptide libraries (Koivunen et al., supra, 1993, 1994); peptidomimetic library (Blondelle et al., Trends Anal. Chem. 14: 83-92 (1995)); oligosaccharide library (York et al., Carb. Res) 285: 99-128 (1996); Liang et al., Science 274: 1520-1522 (1996); and Ding et al., Adv. Expt. Med. Biol. 376: 261-269 (1995)); Protein library (de Kruif et al., FEBS Lett., 399: 232-236 (1996)); glycoprotein or glycolipid library (Karaoglu et al., J. Cell Biol. 130: 567-577 (1995) Or, for example, a chemical library containing drugs or other pharmaceuticals (Gordon et al., J. Med. Chem. 37: 1385-1401 (1994); Ecker an d Crook, Bio / Technology 13: 351-360 (1995), US Pat. No. 5,760,029). A library of diverse molecules can also be obtained from commercial sources.

H. Isolation of hematopoietic progenitor cells The present invention further treats tissue containing HPC with an agent (eg, an antibody) capable of specific binding to integrin α4β1 to isolate the HPC to which the agent binds. To provide a method for isolating HPC from tissue. These methods are based in part on the inventors' discovery that HPC expresses the integrin α4β1.

  In one embodiment, the HPC comprises endothelial progenitor cells (EPC). EPC is useful in controlling angiogenesis (Isner et al., US Pat. No. 5,980,887, incorporated by reference). Heterogeneous, allogeneic and autologous endothelial cell precursor grafts coalesce in vivo to sites of active angiogenesis or vascular injury (i.e., they selectively migrate to such locations) ( Isner et al., US Pat. No. 5,980,887). Endothelial cell precursors are present in many tissues including, for example, peripheral blood, bone marrow and umbilical cord blood. Endothelial cell precursors treat tissue containing endothelial cell precursors (e.g., peripheral blood, bone marrow, umbilical cord blood, etc.) with an antibody capable of specific binding to at least a portion of the integrin α4β1 polypeptide, followed by And can be isolated according to the methods of the invention by isolating cells that bind to the antibody. The endothelial cell precursor properties of the isolated cells are determined by the presence of endothelial cell precursor-specific antigens (e.g., CD34, flk-1, and / or tie-2) on the surface of the isolated cells. This can be confirmed by measuring using a commercially available antibody against the antigens. Although it is not necessary to proliferate endothelial cell precursors in vivo by administering supplemental growth factors (e.g., GM-CSF and IL-3) to patients prior to processing tissue containing endothelial cell precursors May be desirable.

  Thus, in one embodiment, the isolated endothelial cell precursor is pathologically stimulated to promote angiogenesis or an angiogenesis modulator (e.g., an anti-angiogenic or anti-angiogenic agent, respectively). Alternatively, it can be used to deliver to sites of effective angiogenesis. Furthermore, in another embodiment, endothelial cell precursors can be used to reduce restenosis by inducing re-endothelialization of damaged blood vessels and thereby indirectly inhibiting smooth muscle cell proliferation. (Isner et al., US Pat. No. 5,980,887).

  In one preferred embodiment, endothelial cell precursors can be used alone to enhance the patient for angiogenesis. Some patient populations, typically elderly patients, may have either a limited number of endothelial cells or a limited number of functional endothelial cells. Thus, if it is desired to promote angiogenesis, for example to stimulate angiogenesis by using strong angiogenesis such as VEGF, such angiogenesis can be limited by endothelial cell depletion. However, administration of endothelial cell precursors can enhance angiogenesis in those patients.

  Since endothelial cell precursors home to angiogenic lesions, these cells are also useful as autologous vectors for gene therapy and diagnosis of ischemia or vascular injury. For example, these cells can be utilized to inhibit and increase angiogenesis. For example, for anti-tumor therapy, endothelial cell precursors can be transfected with or bound to cytotoxic drugs, cytokines or costimulatory molecules that can stimulate an immune response, other anti-tumor drugs, or angiogenesis inhibitors. Can be done. For the treatment of focal ischemia, angiogenesis can be amplified by pretransfection of endothelial cell precursors to achieve constitutive expression of angiogenic cytokines and / or selected matrix proteins. In addition, endothelial cell precursors can be labeled (eg, radiolabeled), administered to a patient, and used to detect ischemic tissue or vascular damage.

  Successful use of autologous endothelial cell precursor grafts has been shown to be easily manipulated and expanded ex vivo (US Pat. Nos. 5,980,887; 5,199,942; and 5,541,103, The disclosure of which is incorporated by reference).

Once isolated, HPC (such as endothelial progenitor cells) can optionally be stored in a cold state prior to administration to a subject to treat a number of conditions. Administration to a subject can be by any suitable means including, for example, intravenous injection, intravenous bolus, and site-specific delivery by catheter. Preferably, HPC obtained from the subject is re-administered. Generally, about 10 ⁶ to about 10 ¹⁸ HPCs are administered to a subject as a transplant.

  In one embodiment, the HPC (such as endothelial progenitor cells) can be genetically modified or wild type. A “genetically modified” cell refers to a cell that contains a “transgene”, ie, any nucleic acid sequence that is introduced into the cell by experimental manipulation. A transgene can be an “endogenous DNA sequence” or a “heterologous DNA sequence” (ie, “foreign DNA”). Genetically modified cells are contrasted with “wild-type cells” that do not contain a transgene. HPC can be genetic recombination for genes encoding various proteins, including anticancer drugs, as exemplified by genes encoding various hormones, growth factors, enzymes, cytokines, receptors, MHC molecules, and the like. The term “gene” includes both exogenous and endogenous nucleic acid sequences to a cell into which a viral vector, eg, a poxvirus such as a porcine pox containing the human TNF gene, can be introduced. Furthermore, it is interesting to use a gene encoding a polypeptide as a secretion from HPC to give a systemic effect to the protein encoded by the gene. Specific genes of interest include TNF, TGF-α, TGF-β, hemoglobin, interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, Interleukin-7, Interleukin-8, Interleukin-9, Interleukin-9, Interleukin-10, Interleukin-11, Interleukin-12, GM-CSF, G-CSF, M-CSF, human growth Factors, costimulatory factor B7, insulin, factor VIII, factor IX, PDGF, EGF, NGF, EPO, β-globin, cellular mitogens, etc. plus those that encode biologically active modified versions of these proteins Including. A gene can further encode a product that controls the expression of another gene product or blocks one or more steps in a biological pathway. Furthermore, the gene can encode a toxin fused to a polypeptide (eg, a receptor ligand) or an antibody that directs the toxin to a target, such as a tumor cell. Similarly, a gene can encode a therapeutic protein fused to a targeting polypeptide to deliver a therapeutic effect to the affected tissue or organ.

  In another embodiment, HPC (such as endothelial progenitor cells) can also be used to deliver genes that can enhance the ability of the immune system to fight a particular disease or tumor. For example, the cells can be used to deliver one or more cytokines (eg, IL-2) and / or one or more antigens that can boost the immune system.

  In yet another embodiment, HPC (such as endothelial progenitor cells) is also used to selectively administer drugs such as anti-angiogenic compounds such as O-chloroacetylcarbamoyl fumagillol (TNP-470). Can be used. Preferably, the drug is taken up by the cells in a medium such as a liposome, a sustained release capsule or the like. HPC (such as endothelial progenitor cells) selectively targets sites of active angiogenesis, such as rapidly growing tumors, where compounds are then released. This method can reduce undesirable side effects at other locations.

  In further embodiments, HPC (such as endothelial progenitor cells) can be used to enhance angiogenesis in ischemic tissues (ie, tissues that have a blood deficiency as a result of ischemic disease). Such tissues can include, for example, muscle, brain, kidney and lung. The ischemic diseases include, for example, cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, myocardial ischemia. A method for inducing the formation of new blood vessels in ischemic tissue is disclosed in Isner et al., US Pat. No. 5,980,887, which is incorporated herein by reference.

The following examples serve to illustrate certain preferred embodiments and aspects of the invention and should not be construed as limiting its scope.

Example 1
Inhibition of in vivo endothelial progenitor cell migration in mouse and rat animal models Integrin α4β1 inhibitors can be used to prevent endothelial cell precursors from exiting the bloodstream and entering sites of neovascularization. Angiogenesis assays are performed by injecting Matrigel containing angiogenic growth factors, a viscous extracellular matrix that coagulates at body temperature, in mice or nude rats transplanted with mouse Tie2-LacZ bone marrow. Mice are treated by intravenous injection with anti-mouse α4β1 and a control antibody or other inhibitor of α4β1. α4β1 inhibitors are expected to block LacZ stained cells from coalescing into blood vessels, indicating that α4β1 controls exit from the circulation of endothelial progenitor cells. Matrigel cryosections are stained with antibodies directed against CD31 and factor VIII-related antigens to obtain an indication of angiogenesis.

Example 2
Endothelial progenitor cells (EPC) express integrin α4β1 Purified human umbilical vein endothelial cells (`` HUVECS '') (Clonetics, San Diego, Calif.), And circulating CD34 + stem cells (Asahara et al., Science, 275: 964- 967, (1997)), endothelial progenitor cells ("EPC") cultured on fibronectin derived from the mouse, incubated with mouse anti-human integrin α4β1 antibody for 60 minutes on ice, washed twice with PBS, then rhodamine Incubated on ice for 30 minutes in labeled goat anti-mouse IgG. Cells were washed twice with cold PBS and then analyzed with a FACSCAN analyzer for integrin α4β1 expression. The percentage of cells expressing this integrin was measured and plotted by cell type (Figure 13).

  33 percent of endothelial progenitor cells were positive for integrin α4β1 expression, but only 12% of HUVECS were positive. These results indicated that the inhibitory effect of α4β1 antagonists in angiogenesis was due to inhibition of endothelial progenitor cell involvement in angiogenesis.

Example 3
α4β1 antagonist blocks endothelial stem cell contribution to angiogenesis Mouse angiogenesis is the posterior part of 400 μl of Matrigel depleted of growth factors including 400 ng / ml bFGF or VEGF, FVB / N inbred mice They were induced by dorsal flank or by subcutaneous injection into FVB / N mice that had been irradiated and transplanted with bone marrow from Tie2LacZ mice. Animals were treated with 200 μg of endotoxin-free rat anti-mouse α4β1 antibody (PS-2) at 100 μl on day 0 and 3 or control isotype-matched rat anti-mouse integrin on days 1 and 4. Treatment was by intravenous injection of β2 antibody (n = 10). After 5 days, the Matrigel plug was excised, embedded in OCT, frozen, and sectioned. Thin sections (5 μm) were immunostained with rat anti-mouse CD31 followed by Alexa565-conjugated goat anti-rat immunoglobulin. CD31 positive vessel density per 200X microscope field was measured in 5 fields per Matrigel plug. Mean vessel density per field +/- SEM was graphed against processing conditions. Representative field images of control IgG and anti-α4β1-treated bFGF or VEGF-containing plugs stained for β-galactosidase expression were taken, with red indicating CD31 positive vessels and blue indicating the nuclei of all cells. Represent (Figure 14B). Sections from Tie2 / LacZ transplanted mice were analyzed for the presence of bone marrow-derived endothelial cells by staining sections for expression of β-galactosidase using a kit from Life Technologies. For bFGF-stimulated angiogenesis, blue cells in plugs arising from transplanted bone marrow were counted (FIG. 14A).

  Antagonists of the integrin α4β1 prevent the involvement of endothelial progenitor cells in angiogenesis. These mice were irradiated to kill their own bone marrow prior to transplantation of bone marrow from mice expressing LacZ under the endothelium-specific promoter, Tie2 promoter, so that endothelial cells expressing β-galactosidase are , Derived from bone marrow. Thus, endothelial cells originating from the bone marrow turn blue in tissue incubated in β-galactosidase substrate. These data indicated that fewer blue endothelial cells were induced by growth factors in mice treated with anti-α4β1 than in mice treated with control antibody. Therefore, anti-α4β1 inhibited the involvement of bone marrow-derived endothelial progenitors in angiogenesis.

Example 4
Exemplary Materials and Methods The following are some exemplary materials and methods that may be useful in the present invention, particularly in Examples 5-13 and FIGS. 15-20.

A. Chick Chorioallantoic Angiogenesis Assay 10-day-old chick fetal chorioallantoic membrane was tested against 1 μg / ml bFGF and the fibronectin RGD-containing cell binding domain (CBP) and EILDV-containing C-terminal CS-1 domain. And an anti-type-matched control antibody (anti-MHC) was also applied. After 3 days, vessel branch points were counted using 30X magnification. Angiogenesis was stimulated in CAM with 1 μg / ml bFGF, VEGF, TNFα, or IL-8. Antibodies directed against saline or integrin α4β1 (mouse anti-human α4β1 antibodies HP1 / 2, P4G9, P1H4 and rat anti-mouse α4β1 PS2 were all tested and had similar results) and control isotype-matched antibodies. Applied to CAM, vessel branch points were counted after 3 days. Cryosections from bFGF-stimulated saline or antibody treated CAMs were immunostained to detect vascular expression of von Willebrand factor. VWF + structure was quantified in five 200X microscope fields. Each experiment was repeated 3-4 times and results from a representative experiment are shown.

B. Mouse Angiogenesis Assay Angiogenesis was induced in FVB / N mice by subcutaneous injection of 400 μl of growth factor reduced Matrigel supplemented with 400 ng / ml of bFGF or VEGF. Mice were treated on days 0 and 3 by intravenous injection of 200 μg functional blocking rat anti-integrin α4β1 (PS / 2) or isotype matched control antibody (rat anti-integrin β2, BD Pharmingen). The Matrigel plug was excised and stored frozen after 5 days. Cryosections were immunostained to detect CD31 expression and counterstain with DAPI. Microvessel density was quantified in 10 randomly selected 200X microscope fields for each plug in each treatment group (n = 8). Alternatively, angiogenesis was induced in corneal transplantation of polymerized pellets containing 400 ng / ml VEGF in FVB / N mice. Animals (n = 5) were treated with anti-α4β1 (PS / 2) or control IgG on days 0 and 3. Fifteen minutes before sacrifice on day 5, mice were injected intravenously with an endothelium-specific lectin, Bandeira simplifolia-FITC, and the tissues were stored frozen. The angiogenic response to VEGF was quantified as the percent green fluorescent area visible at 100X magnification. Alternatively, 5 million HT29 human α4β1-negative colon cancer cells were implanted subcutaneously in nude mice. If the tumor is palpable (approximately 30 mm ³ ), mice are injected twice weekly with saline, rat anti-mouse α4β1, or isotype-matched control antibody, anti-CD11b integrin (M1 / 70, BD Pharmingen) Processed by. Tumor area was measured every other day and tumor mass was measured 4 weeks after treatment. Mean tumor mass +/- SEM is shown. Cryosections of tumors were immunostained to detect CD31 (BD Pharmingen), and microvessel density was quantified for 5 randomly selected microscopic fields. In addition, tumors were stained with hematoxylin and eosin (n = 10). Three experiments were performed and representative data selected are shown. Statistical significance was determined using Student's t test.

C. FACs analysis Expression profiles of surface antigens on human microvascular endothelial cells, human umbilical vein endothelial cells and endothelial progenitor cells were expressed as human α4β1 (HP1 / 2), αvβ3 (LM609), αvβ5 (P1F6), α5β1 (JBS5), Beta 1 (P4C10), Beta 7 (FIB504, Beckton Dickinson Pharmingen), CD34 (8G12, Becton Dickinson), AC133 (AC133, Miltenyi Biotec), Flk-1 (A-3, Santa Cruz Biotechnology), CD45 (2D1, Beckton Dickinson), CD31 (HEC7, Endogen), VE-cadherin (BV6, Chemicon International) and VCAM (P8B1, Chemicon International) and analyzed by FACs analysis using mouse antibodies directed against rabbit anti-VWF (Dako).

D. Isolation of endothelial progenitor cells Mononuclear cells derived from human peripheral blood were isolated using known methods. In some experiments, CD34 positive cells were purified from a mononuclear population using the MACS magnetic bead system. Cells were cultured for up to 9 days in endothelial growth medium (EGM-2 with 2% fetal bovine serum, bFGF, VEGF). After 7 days, 80% of the cells are spindle-shaped and express vascular cell markers and stem cell markers.

E. Adhesion and migration assay Adhesion was performed essentially as described. For adhesion analysis, EPC from day 7 was added to 5 μg / ml CS-1 fibronectin (recombinant H120 fragment, a kind gift from Martin J. Humphries), recombinant soluble VCAM, plasma fibronectin, vitronectin (as described For 30 minutes to adhere to triplicate wells of a 48 well plate coated with collagen. Plates were washed 5 times and adherent cells were quantified at 200X magnification. Alternatively, cells were stained with crystal violet, washed, air dried and extracted with acetic acid. Absorbance at 600 nm was then measured. In some experiments, 25 μg / ml function blocking antibody to integrin α4β1 (HP1 / 2), α5β1 (JBS5), β1 (P4C10), αvβ5 (P1F6), or αvβ3 (LM609) was added to the adhesion assay. Migration assays were performed as described. EPC was added to triplicate 8 μm pore size transwell inserts coated with 5 μg / ml CS-1 fibronectin (recombinant H120 fragment from Martin J. Humphries), collagen, fibronectin or vitronectin. After 4 hours, the cells were fixed with 3.7% paraformaldehyde, stained with crystal violet, and the cells under the transwell were quantified.

F. Bone marrow transplantation
Bone marrow from Tie2LacZ transgenic mice (n = 8) was transplanted into irradiated FVB / N mice. One month after recovery, angiogenesis was induced by injection of growth factor-reduced matrigel supplemented with 400 ng / ml of bFGF or VEGF. Mice were treated on days 0 and 3 by intravenous injection of 200 μg rat anti-mouse α4β1 antibody (PS / 2) or isotype matched control (anti-b2 integrin). The plugs were excised and stored frozen after 5-7 days. The frozen sections were processed to detect the expression of β-galactosidase in the matrigel plug. Micrographs were taken at 200X and 600X magnification. LacZ positive cells per 200X field were quantified in 10 microscopic fields. Cryosections were also immunostained with rabbit anti-β-galactosidase and rat anti-mouse CD31. Micrographs were taken at 200X. LacZ, CD31 positive vessels were quantified in 10 microscopic fields. Statistical significance was determined using Student's t test.

Example 5
In a study analyzing the role of fibronectin and its receptor in angiogenesis, we found that the RGD cell binding domain of fibronectin and its receptor α5β1 antagonist strongly block angiogenesis (Kim et al., Am J Pathol 2000 Apr; 156 (4): 1345-62). Surprisingly, an antibody that recognizes the EILDV site in the alternatively spliced domain of tissue fibronectin, CS-1 fibronectin, strongly blocked angiogenesis in the chicken chorioallantoic membrane (CAM) model (Figure 15A, P <0.05). These antagonists interfere with the binding of CS-1 fibronectin to its principal receptor, integrin α4β1, (Guan et al., Cell 1990 Jan 12; 60 (1): 53-61). Importantly, these antibodies inhibit the adhesion and migration of human endothelial cells cultured on CS-1 fibronectin. These results suggest that CS-1 fibronectin and its receptor α4β1 may play a role in angiogenesis. These findings indicate that our previous observation that fibronectin expression is significantly up-regulated during angiogenesis (Kim et al. 2000, supra), and that CS-1 fibronectin expression is skin, heart and other A third-party report showing upregulation in relation to blood vessels during wound repair in other tissues, as well as in chronic inflammatory diseases such as rheumatoid arthritis (Elices et al., J Clin Invest 1994 93 (1 ): 405-16; Morales-Ducret et al., J Immunol 1992 149 (4): 1424-31). Based on these results, we considered whether α4β1 is involved in angiogenesis. To evaluate the role of α4β1 in angiogenesis, α4β1 function blocking antibodies were applied to CAMs stimulated with bFGF, VEGF, TNFα or IL-8. Anti-α4β1 blocked angiogenesis induced by each of these growth factors (FIG. 15B, P <0.05). These studies indicate that the integrin α4β1 plays a role in neovascularization in the chicken CAM model.

Example 6
To assess the role of α4β1 in mammalian angiogenesis, the effects of integrin α4β1 antagonists were tested in several mouse models of neovascularization. Anti-integrin α4β1 antibody (PS / 2) was injected intravenously into mice stimulated to undergo angiogenesis by subcutaneous injection of saturated matrigel of either bFGF or VEGF (FIG. 15C). We have found that inhibition of α4β1 significantly blocks angiogenesis, whether assessed by microvessel density or by total vessel volume (P <0.05). In addition, a peptide antagonist of α4β1 (EILDV, derived from CS-1 fibronectin) also blocked neovascularization in this model and provided further support for the role of α4β1 in this process. Anti-α4β1 antibody also blocked corneal neovascularization (P <0.05). Importantly, antagonists of integrin α4β1 significantly inhibited tumor angiogenesis and tumor growth (FIGS. 15D-E). Thus, CS-1 fibronectin and its receptor integrin α4β1 play an important role in the control of neovascularization.

Example 7
The inventors next inferred that if α4β1 controls angiogenesis, this integrin should be expressed on tumor vasculature and other neovascular tissues. In order to evaluate the expression pattern of integrin α4β1 on the neovascular bed of human tumors, we performed immunohistochemistry to detect the expression of integrin α4β1 and von Willebrand factor, a marker of vascular endothelium. (Kim et al. 2000, supra). Using various monoclonal anti-α4 antibodies, we were surprisingly unable to show α4 expression on the tumor endothelium, but control tissues such as lymph nodes and human melanoma Integrin α4β1 expression was detected in (see; FIG. 16A). Occasionally, we detected α4β1 expression on a subset of blood vessels within the tumor, such as invasive ductal carcinoma (FIG. 16A). Using antibodies that react with the cytoplasmic tail of α4, we have developed high levels of α4 on vascular endothelial cells in growth factor-stimulated CAM, growth factor-stimulated mouse tissue, mouse tumors and human tumors. Expression could be detected (FIG. 16B). However, unlike the new blood vessel integrins α5β1 and αvβ3 (Brooks et al., Science 1994 264: 569-71), integrin α4β1 is weakly expressed on proliferating human microvessels or venous endothelial cells in vitro Only (FIG. 16C).

Example 8
Since α4β1 is only weakly expressed on proliferative purified endothelial cells, we have found that this integrin is transiently expressed by endothelial cells in vivo or during neovascularization It was inferred that it may be expressed by endothelial precursors. New blood vessels can be generated not only by budding, but also by inoculation of bone marrow-derived stem cells in tissues, and we considered whether EPCs express α4β1. To isolate EPC, a mononuclear fraction of peripheral blood leukocytes or CD34 positive stem cells (isolated from the mononuclear fraction of peripheral blood leukocytes) on a fibronectin culture plate in the presence of the angiogenic cytokines bFGF and VEGF In culture. The resulting EPCs not only express high levels of α4β1, but also CD34, CD133, Flk-1 (Asahara et al., 1997 Feb supra; Brooks et al., Science 1994 supra), CD45 and CD18. Stem cell markers as well as endothelial markers such as VE-cadherin, VCAM, CD31 and VWF are co-expressed (FIGS. 16D-E). As the time of culture increases, these cells acquire additional features of endothelial cells and express increasing amounts of CD34, VE-cadherin, VCAM and VWF (FIGS. 16D-E). These cells show larger, elongated, sticky morphology and spontaneously form tube-like structures (FIG. 16F). Importantly, EPC is strongly positive for integrin α4β1, but is unable to express the closely related leukocyte integrin α4β7 (FIG. 16D). EPC is also positive for other adhesion receptors including integrin α5β1, RGD-binding fibronectin receptor (Kim et al. 2000, supra). Significantly, EPCs expressed little integrin αvβ5 and no αvβ3 early in the development in vitro, but at later stages high levels of these integrins were observed (Figure 16E), these integrins were It suggests that EPC is upregulated as it acquires more and more endothelial features. EPC is also positive for UEA lectin staining, endothelial cell characteristics, and binds DiI acetylated LDL. Thus, in contrast to mature endothelial cells, EPC is strongly positive for integrin α4β1 expression.

Example 9
The integrin on circulating lymphocytes is often maintained in an inactive or low affinity state (Peichev et al., Blood 2000 Feb 1; 95 (3): 952-8). Et al. Next determined whether the integrin α4β1 expressed by EPC could function. Indeed, EPCs adhere to and migrate on CS-1 fibronectin, plus collagen, plasma fibronectin and vitronectin. Importantly, adhesion to CS-1 fibronectin is mediated by integrin α4β1, but function-blocking anti-α4β1 and β1 antibodies prevent EPC from adhering to this matrix protein (FIG. 17A). Since α4β1 is also a receptor for the immunoglobulin superfamily molecule VCAM expressed by activated endothelium, we also have the ability of EPC to adhere to plates coated with recombinant soluble VCAM (rsVCAM) I investigated. An antagonist of α4β1 blocked EPC adhesion to VCAM, indicating that integrin α4β1 is a functionally active receptor for both CS-1 fibronectin and VCAM on endothelial stem cells (FIG. 17B). .

Example 10
To determine whether EPC can adhere to proliferating vascular endothelium stimulated by angiogenic growth factors, EPC labeled with DiI acetylated LDL was placed on a proliferative endothelial monolayer. EPC strongly bound to the endothelium in an α4β1-dependent manner (FIG. 17C), and rsVCAM blocked EPC adhesion to the endothelial monolayer (FIG. 17D). Similar results were obtained when α4β1 antibody or rsVCAM were preincubated with EPC, but not when they were preincubated with endothelial monolayers. Thus, α4β1 mediates adhesion of EPC to VCAM on the vascular endothelium. VCAM is upregulated in the angiogenesis endothelium in response to inflammatory cytokines and growth factors, and the recombinant soluble form of VCAM can inhibit angiogenesis (Nakao et al., J. Immunol. 2003 Un 1; 170 (11): 5704-11), these results show that α4-VCAM interactions can promote migration of bone marrow-derived stem cells or progenitor cells into tissues during angiogenesis and tissue repair It suggests that there is sex. Indeed, the integrin α4β1-VCAM interaction is in vivo during fetal development (chorionic-allantoic, endocardial-myocardial, primary myoblast fusion), in immune cell transport (lymphocytes and monocytes in inflammation) Plays an inevitable role in promoting heterotypic cell adhesion in the retention of immune cell precursors in the bone marrow (Rosen et al., Cell 1992 Jun 26; 69 ( 7): 1107-19). Thus, α4-VCAM interaction can control the entry of stem cells into the site of tissue repair.

Example 11
To determine whether α4β1 controls the formation of new blood vessels by EPC, DiI-labeled human EPC and anti-human α4β1 or control antibodies in Matrigel containing VEGF were implanted subcutaneously into nude mice. After 5 days, new blood vessels were visualized with Bandeira simplifolia injection. We observed that many EPCs formed blood vessels (FIG. 18A) and that they were not control antibodies, but anti-α4β1 antibodies blocked angiogenesis. These studies indicate that EPC is capable of forming new blood vessels and that α4β1 function is required for this process.

Example 12
To determine whether α4β1 mediates EPC adhesion to angiogenic endothelium in vivo, human EPCs were adoptively transferred to nude mice with integrin α4β1-negative colon cancer tumors implanted subcutaneously. Mice were treated systemically with anti-human α4β1 antibody, control antibody or saline. We found that EPCs merged into new blood vessels and that only an antagonist of human α4β1 blocked this event (FIG. 18B). These findings demonstrate that integrin α4β1 mediates extravasation from endothelial stem cell circulation to angiogenic tissue. These studies suggest that α4β1 mediates stem cell transport in vivo because all CD34 positive stem cells must pass through the endothelium to enter the tissue.

Example 13
To investigate the role of α4β1 in the regulation of bone marrow-derived endothelial stem cell transport in vivo, angiogenesis was induced in mice transplanted with bone marrow from Tie2LacZ mice and the animals were treated with anti-mouse α4β1 antibody (PS / 2) And systemically treated with a control antibody (anti-mouse β2 integrin). It was determined that anti-α4β1 antibody but not anti-β2 antibody significantly blocked the combination of LacZ positive cells and blood vessels in Matrigel, whether angiogenesis was induced by bFGF or VEGF (Figures 18C-D). ). To further determine whether LacZ positive cells merged into blood vessels and expressed endothelial markers, frozen sections were immunostained with anti-β-galactosidase (green) and anti-mouse CD31 (red). A significant number (> 90%) of LacZ-positive bone marrow-derived cells were identified in CD31-positive blood vessels and observed that antagonists of α4β1 blocked the coalescence of these cells into new blood vessels (FIGS. 18E-F). . These studies show that α4β1 promotes the influx of bone marrow-derived endothelial stem cells into tissues involved in the formation of new blood vessel structures.

Example 14
Additional data herein is shown in FIG. FIG. 19A shows endothelial cell migration on 8 μm pore transwells coated with 5 μg / ml CS-1 fibronectin in the presence of medium, anti-CS-1 fibronectin or control antibody (W6 / 32, anti-MHC). Is shown. FIGS. 19B, C show endothelial cell adhesion to plastic plates coated with 5 μg / ml CS-1 fibronectin in the presence of medium, anti-α4β1 (HP1 / 2) or control antibody (P1F6). FIG. 19D shows that frozen sections from saline or antibody-treated CAMs stimulated with bFGF were immunostained to detect vascular expression of von Willebrand factor. VWF + structure was quantified in five 200X microscope fields. FIG. 19E shows that angiogenesis was induced in corneal transplantation of polymerized pellets containing 400 ng / ml VEGF in FVB / N mice. Animals (n = 5) were treated with anti-α4β1 (PS / 2) or control IgG (cIgG) on days 0 and 3. Fifteen minutes before sacrifice on day 5, mice were injected intravenously with an endothelium-specific lectin, Bandeira simplifolia-FITC, and the tissues were stored frozen. The angiogenic response to VEGF was quantified as the percent green fluorescence area visible under high magnification (100X). FIGS. 19F-G show 400 ng / ml bFGF containing (F) 200 μg function-blocking rat anti-integrin α4β1 (PS / 2) or isotype matched control antibody (rat anti-integrin β2) in nude mice. FIG. 19G shows the use of 50 μM EILDV or EILEV peptide, which was induced by subcutaneous injection of supplemented 400 μl growth factor reduced Matrigel. Mice were injected intravenously with the endothelium-specific lectin, Bandeira simplifolia-FITC, 15 minutes prior to sacrifice on day 5. Matrigel plugs were homogenized in RIPA buffer and the fluorescence intensity was measured.

Example 15
Additional data herein is shown in FIG. FIG. 20A shows EC, EPC and fibroblast cell fluorescence analysis for UEA-1 lectin binding and DiI-acetylated LDL uptake. FIG. 20B shows the adhesion of purified EPC to plastic plates coated with 5 μg / ml fibronectin, CS-1 fibronectin, vitronectin and collagen. FIG.20C shows the migration of purified EPC on 8 μm pore transwells coated with 5 μg / ml fibronectin, CS-1 fibronectin, vitronectin and collagen, and FIG.20D shows the medium, anti-α4β1 (HP1 / 2 ), Anti-αvβ3 (LM609), anti-αvβ5 (P1F6), or anti-α5β1 (P1F6) in the presence of purified EPCs on plastic plates coated with 5 μg / ml vitronectin.

Example 16
The following are some exemplary materials and methods that may be useful in the present invention, particularly in Examples 17-24 and FIGS. 33-36. Statistical significance was determined using Student's t test.

A. Stem cell isolation:
3.7 × 10 ⁹ mononuclear cells were purified by Histopaque gradient centrifugation from 6 units of human buffy coat from San Diego Blood Bank. CD34 cell isolation was performed by positive selection through two anti-CD34 columns using a kit from Miltenyi Biotech (Auburn, CA). The yield of CD34 + cells was 3 × 10 ⁶ cells with 89% purity as assessed by FACs analysis.

B. In vivo microscopy Stem cells were labeled with 5- and -6-4-chloromethylbenzoylamino-tetramethyl-rhodamine (CMTMR, Invitrogen, Carlsbad, Calif.) For 15 minutes on ice in medium and washed. 1 × 10 ⁶ labeled stem cells were injected intravenously into mice with N202 syngeneic GFP-expressing tumor spheroids grown on transplanted mammary fat pads under transparent dorsal subcutaneous fat chambers. Animals were sedated (15-20 minutes) and at the same time in vivo fluorescence microscopy was performed using an epi-illuminator and a video-triggered stroboscopic illumination (MV-7600, EG & G, Salem, MA) from Xenon Arc Performed using an Instrument Microscope (Mikron Instrument, San Diego, CA). A silicon multiplication target camera (SIT68, Dage-MTI, Michigan City, IN) is attached to the microscope. A Hamamatsu image processor (Argus 20) containing firmware version 2.50 (Hamamatsu Photonic System, USA) is used for image processing and for capturing images into a computer. A Zeiss Achroplan 20X / 0.5 W objective lens 10 / 0.22 was used to capture the image.

C. FACs analysis
FACs analysis was performed at the UCSD Cancer Center Core facility. Expression of integrin α4β1, CD34, and CD133 on stem cells is expressed in PE-conjugated mouse anti-human α4β1 (HP2 / 1, Chemicon International, Temecula, Calif.), FITC and PE-conjugated mouse anti-human CD34 (AC136, Miltenyi Biotech, Auburn , CA), and PE-conjugated CD133 (AC133 / 1, Miltenyi Biotech, Auburn, Calif.), CD31 (HEC7, Pierce), and two-color fluorescence. VCAM expression on EC was measured with P8B1 (Chemicon International, Temecula, CA).

D. Immunohistochemistry Cryosections were fixed in cold acetone for 2 minutes, air dried and rehydrated in phosphate buffered saline (PBS) for 5 minutes. Slides were washed in 0.05-0.1% Triton X-100 in PBS for 2 min, 2% in 5% bovine serum albumin in PBS overnight at 4 ° C. and 2 in primary antibody (5-10 μg / ml). Incubated at RT for 3 hours, washed 3 times in PBS and incubated at RT in secondary antibody at 1 μg / ml for 1 hour. Slides were washed 3 times in PBS, stained with DAPI and covered with coverslips. The primary antibodies are: fibronectin (TV-1, Chemicon), anti-mouse VCAM (M / K-2 from Chemicon), anti-pan species VCAM (H-276 from Santa Cruz, sc -8304), and anti-mouse CD31 (MEC 13.3 from Pharmingen).

E. Adhesion assay Adhesion assays were performed as described (Kim et al. (2000) Am. J. Pathol. 156, 1345-1362) and recombinant H120 CS-1 fibronectin (Martin J. Humphries, University of (From Birmingham, UK) on plastic 48-well plates coated with 5 μg / ml. Stem cells were incubated for 30 minutes on the coated plates in the presence of 25 μg / ml anti-α4β1_ (HP2 / 1) or anti-αvβ5 (P1F6, Dr. David Cheresh, the Scripps Research Institute). CMTMR-labeled stem cells were incubated for 60 minutes at 37 ° C. on HUVEC monolayers in the presence of 25 μg / ml HP2 / 1 or P1F6 in HEPES buffered serum-free medium. Unbound cells were removed by gentle washing with PBS. Cells were fixed in 3.7% paraformaldehyde. A representative field was photographed at 200X and the number of cells adhering per field was quantified in five representative fields for each treatment condition.

F. Adoptive transfer tumor research
3X10 ⁶ CMTMR-labeled stem cells were added to saline, 50 μg / ml control antibody (LM609, anti-human αvβ3) or anti-human α4β1 (HP2 / 1, gift from Roy Lobb, Biogen, or 9F10, Becton-Dickinson, (San Diego, CA). Cells were incubated with antibodies for 30 minutes on ice prior to injection into nude mice bearing N202 or Lewis lung cancer tumors. One hour later, the animals were sacrificed. Tumors with surrounding connective tissue added were excised and stored frozen (n = 6).

  Alternatively, lineage negative (Lin-) cells were isolated from bone marrow of EGFP mice by negative immunoselection as previously described (Otani et al. (2002) Nat. Med. 8, 1004-1010). . Cells were injected into nude mice with 0.5 cm Lewis lung cancer tumors. Animals were treated with saline, control antibody (anti-CD11b) or anti-mouse α4β1 antibody (PS / 2) for the next 5 days. After 5 days, the animals were sacrificed. Tumors with surrounding connective tissue added were excised and stored frozen (n = 6).

G. Bone marrow transplantation
Bone marrow from FVB / N-Tie2LacZ mice was transplanted into irradiated FVB / N mice. After 12 weeks, mice were injected with 400 μl growth factor-reduced matrigel supplemented with 400 ng / ml bFGF or VEGF, and on days 1 and 3, 200 μg / mouse rat anti-mouse α4β1 antibody (low endotoxin) PS / 2, a kind gift from Biogen) or rat anti-mouse β2 integrin (low endotoxin M1 / 70, Becton-Dickinson, San Diego, Calif.). Plugs were removed after 5 days (n = 8). The study was conducted twice. Cryosections were incubated in X-gal or immunostained with rabbit anti-β-galactosidase and rat anti-mouse CD31 (MEC13.3, Becton-Dickinson, San Diego, Calif.). Positive blood vessels were quantified at 200X in 10 randomly selected microscopic fields.

Example 17
Stem cells selectively home to new blood vessel structures Real-time in vivo microscopy that can study the migration of stem cells transplanted into mice with breast cancer to understand how stem cells home to new blood vessel structures Was used. Human CD34 + stem cells were labeled with red fluorescent cell tracking dye and injected into the circulation of nude mice transplanted with mouse mammary gland spheres on the mammary fat pad under the dorsal subcutaneous fat chamber (FIG. 33a). In vivo microscopy was performed immediately thereafter to follow the homing of the stem cells to the tumor. Tumors and associated non-fluorescent blood vessels were visible (Figure 33b), allowing cell adhesion within the vasculature to be assessed. Circulating fluorescent cells were evident in both central and peripheral tumor vasculature, but they stopped only in blood vessels around the tumor (FIGS. 33c-d). In contrast, stem cells rarely stopped at the tumor center (FIGS. 33c-d) or in other organs. Tumor postmortem analysis by fluorescence microscopy showed that stem cells (red, arrowheads) were identified only by anti-CD31 immunostaining (green, arrows), stopped only in tumor peripheral vasculature, or extravasated to adjacent tissues Was confirmed (FIG. 33e). These studies show that stem cells selectively home to increasing peripheral tumor vasculature, suggesting that specific cell adhesion mechanisms may play a role in this homing response.

Example 18
Stem cells express integrin α4β1 To determine how stem cells arrest in peripheral vasculature, the role of cell adhesion molecules in stem cell homing was investigated. Circulating cells such as lymphocytes utilize integrin α4β1 to arrest on the endothelium and to extravasate from the circulation (Guan et al. (1990) Cell 60, 53-61; and Elices et al. al. (1990) Cell 60, 577-584), hematopoietic progenitor cells use α4β1 to adhere to the bone marrow endothelium (Simmons et al. (1992) Blood 80, 388-395; Papayannopoulou et al. (2001). Blood 98, 2403-2411; Craddock et al. (1997) Blood 90, 4779-4788; Miyake et al. (1991) J. Cell Biol. 114, 557-565). To evaluate the role for α4β1 in stem cell homing, the expression of α4β1 on circulating stem cells was examined by FACs analysis. The majority of CD34 + cells express α4β1 and can differentiate into the endothelium (Peichev et al. (2000) Blood 95, 952-958), and virtually all of the CD34 + CD133 + subsets are integrin It was found that α4β1 was expressed (FIG. 34a).

Example 19
Integrin α4β1 is a functionally active receptor in stem cells Integrins in circulating cells are often maintained in an inactive or low affinity state (Bartolome et al. (2003) Mol. Biol. Cell 14, 54 -66) Next, it was determined whether α4β1 on stem cells had functional activity. The integrin α4β1 is a cellular fibronectin (CS-1 fibronectin) (Elices et al. (1994) J. Clin. Invest. 93, 405-416), and VCAM, an immunity expressed on endothelium in inflammatory tissues It is the receptor for the globulin superfamily molecule (Elices et al. (1990) Cell 60, 577-584). Stem cells readily adhered to CS-1 fibronectin-coated plates; this adhesion was blocked by anti-α4β1 antibody but not control (anti-αvβ5) antibody (FIG. 34b). These results indicate that the integrin α4β1 is a functionally active receptor on many stem cells.

Example 20
Interaction of integrin α4β1 with VCAM and / or fibronectin mediates adhesion of stem cells to endothelial cells in vitro Determines whether stem cells can adhere to endothelial cells (EC) in an α4β1-dependent manner For this, fluorescently labeled stem cells were placed on confluent EC monolayers expressing the α4β1 ligand VCAM (FIG. 34c). Stem cells bound strongly to EC (FIGS. 34d-e). This adhesion was blocked by an antibody antagonist of α4β1, but not by a control antibody (anti-αvβ5) (FIGS. 34d-e). Adhesion was also blocked by recombinant soluble VCAM, a competitive inhibitor of integrin α4β1 function. These studies demonstrate that α4β1 can mediate stem cell adhesion to ECs in vitro, and that α4-VCAM or α4-fibronectin interactions promote stem cell adhesion to vasculature. It suggests that you can.

Example 21
Neovascular structural cells express integrin α4β1 ligand VCAM and fibronectin in vitro Next, whether the tissue undergoing neovascularization expresses α4β1 ligand VCAM and cellular fibronectin is examined for these molecules for mouse breast cancer Or examined by examining normal tissue. Both molecules (green) are expressed on the tumor endothelium (red, FIG. 35a) at a much higher level in the periphery of the tumor than in its center (FIG. 35a). Although these ligands are rarely expressed by normal endothelium, fibronectin was occasionally observed around large blood vessels (FIG. 35a). These results demonstrate that the α4β1 ligand VCAM and fibronectin are in the correct position to promote α4β1 + stem cell adhesion.

Example 22
The integrin α4β1 antibody is fluorescently labeled to determine whether α4β1 mediates adhesion of stem cells to proliferating blood vessels in vivo, which inhibits migration of stem cells to new vasculature in vivo Stem cells were introduced by tail vein injection into nude mice with established mouse breast cancer (N202) or Lewis lung cancer (LLC) tumors. Tissues were removed for analysis within 1 hour of cell injection. Stem cells (red) arrested or extravasated in or near blood vessels of both tumor types (green) (FIG. 35b). Significantly, when stem cells were co-injected with a function-blocking anti-human α4β1 antibody, they were unable to arrest in the vasculature of either tumor type (FIGS. 35b-d). In contrast, saline or control antibody had minimal effect on stem cell arrest and adhesion (FIGS. 35b-d). Although stem cells homed to tumor vasculature, they did not home to adjacent normal tissue or other organs such as the lung. These studies indicate that α4β1 regulates homing of stem cells to tumor neovasculature. These results discount non-specific homing of cells to leaky tumor blood vessels because stem cells do not stay in the central tumor blood vessels and antagonists of certain receptors block their adhesion in the blood vessels.

Example 23
The integrin α4β1 antibody inhibits the differentiation of stem cells into vascular endothelium in vivo. Protein) Lineage negative (Lin-) bone marrow derived cells from mouse ⁶ were injected into animals with LLC tumors in the presence or absence of α4β1 antagonist and control antibody. After 5 days, it was found in vivo that EGFP + cells in control-treated animals homed to tumors in significant numbers (arrowheads) and formed EGFP + blood vessels (arrows, FIGS. 36a-b). In contrast, only a few EGFP + cells and no EGFP + blood vessels were observed in the tumors of anti-α4β1-treated mice (FIGS. 36a-b). These studies have shown that blocking homing of stem cells into tumor vasculature inhibits their differentiation into vascular endothelium.

Example 24
The integrin α4β1 antibody inhibits migration of bone marrow stem cells to new blood vessel structures in vivo The above data suggested that integrin α4β1 mediates homing of stem cells directly from bone marrow to tumors. To evaluate this, mice were transplanted with bone marrow from Tie2LacZ mice. In these mice, bone marrow-derived cells that differentiate into endothelium express β-galactosidase in vivo under the control of the promoter of endothelial protein Tie2. Angiogenesis was stimulated in these mice by implantation of growth factor-reduced matrigel saturated with VEGF or bFGF. These growth factors induced an angiogenic response and homing of β-galactosidase positive cells (FIGS. 36c-d). Treatment with mouse anti-α4β1 antibody completely blocked the incorporation of β-galactosidase positive cells into Matrigel, but not anti-β2 integrin antibody (FIGS. 36c-d). In control treated animals, the majority of β-galactosidase positive cells coalesced into new blood vessel structures (arrows) as measured by anti-CD31 (red) and anti-β-galactosidase (green) immunostaining (Figure 36e). ~ F). Importantly, antagonists of α4β1 completely blocked this coalescence (FIGS. 36e-f). Taken together, these studies show that β2 integrin does not, but α4β1 enhances stem cell trafficking by promoting their adhesion to new vascular structures in remodeling tissues.

The polypeptide sequence of human α4 subunit (SEQ ID NO: 1), GenBank accession number XP_039012.1 is shown. The polypeptide sequence of human β1 subunit (SEQ ID NO: 2), GenBank accession number P05556, is shown. The polypeptide sequence of human vascular cell adhesion molecule (VCAM), GenBank Accession No. P19320 (SEQ ID NO: 3) (A) and XP_035774 (SEQ ID NO: 96) (B) are shown. The polypeptide sequence of human fibronectin (SEQ ID NO: 4), GenBank accession number P02751, is shown. 2 illustrates exemplary agents that inhibit integrin α4β1 binding to VCAM. An exemplary agent that inhibits the binding of integrin α4β1 to its ligand is shown and IC50 values are based on a direct binding assay. In this figure, abbreviations are as follows: FCA, 9-fluorene carboxyl; IC, inhibitory concentration; PA, phenylacetyl. FIG. 3 shows an exemplary β-turn mimetic that inhibits integrin α4β1 binding to fibronectin. 1 shows the cDNA sequence of human integrin α4 subunit cDNA (SEQ ID NO: 5), GenBank accession number XM — 039012. 1 shows the cDNA sequence of human integrin α4 subunit cDNA (SEQ ID NO: 6), GenBank accession number XM — 039012. The cDNA sequence of human integrin β1 subunit cDNA (SEQ ID NO: 7), GenBank accession number X07979, is shown. The human VCAM cDNA sequence (SEQ ID NO: 8), GenBank accession number X53051, is shown. The sequence of human fibronectin cDNA (SEQ ID NO: 9), GenBank accession number X02761, is shown. 2 shows a graph of the percentage of cells expressing integrin α4β1 versus human umbilical vein endothelial cells (HUVEC) and endothelial progenitor cells (EPC). A graph of the number of β-galactosidase positive cells per 100X field for antibody treatment (panel A) and a photograph of an immunostained frozen section of excised Matrigel plugs (panel B) is shown. Figure 2 shows that integrin α4β1 and CS-1 fibronectin control angiogenesis. (A) Vascular bifurcation at 30X magnification in CAM stimulated with 1 μg / ml bFGF and treated with anti-fibronectin CBP or CS-1 function blocking antibody or control antibody. (B) Vascular branch point in bFGF, VEGF, TNFα or IL-8 stimulated CAM treated with saline, anti-integrin α4β1 (HP1 / 2) or control isotype matched antibody. (C) Angiogenesis was induced in FVB / N mice by subcutaneous injection of growth factor-reduced matrigel supplemented with bFGF or VEGF, and mice (n = 8) were rat anti-integrin α4β1 (PS / 2) (white Bars) or isotype-matched control antibody (rat anti-integrin β2) (black bars). Microvessel density was quantified at 200X by CD31 immunohistochemistry (right). (D-E) HT29 human α4β1-negative colon cancer cells were implanted subcutaneously in nude mice (n = 10) and mice with palpable tumors (approximately 30 mm ³ ) were treated with saline, rat anti-mouse α4β1. Or an isotype-matched control antibody, anti-CD11b integrin, was treated by intravenous injection. (D) Average tumor mass +/− SEM. (E) CD31 positive microvessels were detected by immunohistochemistry and quantified per 200X field. Shown is the expression of α4β1 on endothelial cells and endothelial progenitor cells. (A) 5 micron thick frozen sections of human lymph nodes, melanoma, and invasive ductal carcinoma were immunostained with anti-vWF (green) antibody and P1H4, anti-human α4β1 (red) antibody. (B) Human breast cancer, mouse MTAG spontaneous breast cancer, mouse VEGF Matrigel, normal mouse liver, bFGF-stimulated CAM, and normal CAM 5 micron thick frozen sections were prepared with anti-vWF (red) antibody and goat anti-α4 cytoplasm Immunostained with tail (green) antibody. Expression consistency is represented in yellow. C) HMVEC FACs analysis for CD31 and α4β1 expression. (D) FACs analysis of expression levels in EPCs on days 4 and 7 for CD34, AC133, and Flk-1. (E) FACs analysis of expression levels in EPC on days 4 and 7 for CD31, VE-cadherin, VCAM and VWF. (F) FACs analysis of expression levels in EPC on days 4 and 7 for β1, β7, β2, α4β1, αvβ3, αvβ5 and α5β1. (G) Photomicrograph of EPC under transmitted light on day 4, 7 or 9 during culture. The functional role of α4β1 expressed in EPC is shown. (A) on a plastic plate coated with (A) 5 μg / ml CS-1 fibronectin or (B) recombinant soluble VCAM in the presence of medium, anti-α4β1 (HP1 / 2) or control antibody (P1F6) Adhesion of purified EPC to. (C) Adhesion of DiI labeled purified EPC to HUVEC monolayer VCAM in the presence of medium, anti-α4β1 (HP1 / 2) or control antibody (P1F6). Cells were quantified by counting adherent red fluorescent cells per 200X microscope field. (D) Adhesion of DiI labeled purified EPC to HUVEC monolayer in the presence of medium, rsVCAM or control protein. Statistical significance was determined using Student's t test. We show that the integrin α4β1 regulates endothelial progenitor cell traffic in vivo. (A) DiI acetylated-LDL (red) labeled endothelial progenitor cells were grown in matrigel depleted of growth factors at 400 ng / ml VEGF, no antibody, control antibody (P1F6) or anti-human α4β1 antibody (HP1 / Mixed with 2). Five days later, the mice were injected with Bandeira simplicifolia lectin-FITC (green) and sacrificed. Cryosections were analyzed by fluorescence microscopy. (B) DiI acetylated-LDL-labeled endothelial progenitor cells are injected intravenously with no antibody, control antibody (P1F6), or anti-human α4β1 antibody (HP1 / 2) into animals with 200 mm ³ HT29 colon cancer tumors It was done. Five days later, the mice were injected with Bandeira simplicifolia lectin-FITC and sacrificed. Cryosections were analyzed by fluorescence microscopy. (CD) Tie2LacZ positive bone marrow was transplanted into irradiated FVB / N mice. After a 1 month recovery period, angiogenesis was induced with bFGF (B) or VEGF (C) and mice were injected intravenously with rat anti-mouse α4β1 (PS / 2) or isotype-matched control (anti-b2 integrin) antibodies. Processed (n = 8). Cryosections were processed to detect the expression of β-galactosidase in Matrigel plug (200X); inset, 600X. The average +/- SEM of LacZ positive cells per 200X field was measured. (E) LacZ positive cells were detected in blood vessels at 200X by immunostaining for β-galactosidase (green) and CD31 (red) expression. Blood vessels that are positive for both are yellow (arrows). (F) Mean +/− SEM LacZ + CD31 + vessels (n = 8). Statistical significance was determined using Student's t test. (A) Endothelial cell migration on 8 μm pore transwells coated with 5 μg / ml CS-1 fibronectin in the presence of medium, anti-CS-1 fibronectin, or control antibody (W6 / 32, anti-MHC) Indicates. (B, C) Adhesion of endothelial cells to plastic plates coated with 5 μg / ml CS-1 fibronectin in the presence of medium, anti-α4β1 (HP1 / 2), or control antibody (P1F6). (D) Saline or antibody-treated CAMs stimulated with bFGF were immunostained to detect vascular expression of von Willebrand factor. (E) Angiogenesis was induced in corneal transplantation of polymerized pellets containing 400 ng / ml VEGF in FVB / N mice. Animals (n = 5) were treated with anti-α4β1 (PS / 2) or control IgG (cIgG) on days 0 and 3. Fifteen minutes before sacrifice on day 5, mice were injected intravenously with an endothelium-specific lectin, Bandeira simplifolia-FITC, and the tissues were stored frozen. The angiogenic response to VEGF was quantified as the percent green fluorescence area visible under high magnification (100X). (F to G) Angiogenesis is performed in nude mice with (F) 200 μg function-blocking rat anti-integrin α4β1 (PS / 2) or isotype-matched control antibody (rat anti-integrin β2), and (G) 50 μM EILDV or EILEV peptide. Elicited by subcutaneous injection of 400 μl growth factor reduced Matrigel supplemented with 400 ng / ml bFGF. Mice were injected intravenously with the endothelium-specific lectin, Bandeira simplifolia-FITC, 15 minutes prior to sacrifice on day 5. Matrigel plugs were homogenized in RIPA buffer and the fluorescence intensity was measured. (A) Cell fluorescence analysis for UEA-1 lectin binding and DiI-acetylated LDL uptake of EC, EPC and fibroblasts. (B) Adhesion of purified EPC to plastic plates coated with 5 μg / ml fibronectin, CS-1 fibronectin, vitronectin and collagen. (C) Migration of purified EPC on 8 μm pore transwells coated with 5 μg / ml fibronectin, CS-1 fibronectin, vitronectin and collagen. (D, E) on a plastic plate coated with 5 μg / ml vitronectin in the presence of medium, anti-α4β1 (HP1 / 2), anti-αvβ3 (LM609), anti-αvβ5 (P1F6), or anti-α5β1 (P1F6) Adhesion of purified EPC. It shows that bone marrow-derived cells can differentiate into endothelial cells (EC). The integrin α4β1 is shown to be an early marker of endothelial progenitor cells (EPC). Endothelial progenitor cells remain integrin α4β1 positive, indicating that they acquire αv integrin expression. Endothelial progenitor cell maturation on days 4, 7 and 9 is shown. We show that integrin α4β1 mediates adhesion of endothelial progenitor cells to fibronectin. We show that integrin α4β1 mediates adhesion of endothelial progenitor cells to rsVCAM. It shows that endothelial progenitor cells adhere to the endothelial monolayer via integrin α4β1. It shows that endothelial progenitor cells adhere to the endothelial monolayer in a VCAM-dependent manner. 2 shows an exemplary Tie2BMT model for the role of hematopoietic stem cells in neovascularization. An antagonist of integrin α4β1 is shown to block the influx of endothelial progenitor cells into the new vascular bed. Integrin α4β1 is shown to promote the contribution of endothelial progenitor cells to angiogenesis in vivo. We show that the integrin α4β1 promotes the involvement of endothelial progenitor cells in extravasation and angiogenesis. We show that human CD34 + stem cells home to peripheral tumor vasculature. (a) CMTMR-labeled stem cells were injected into nude mice with breast cancer under the dorsal skinfold transparent chamber. (b) Tumor and vascular structure in the upper, clear tuft. The lower, peripheral and central tumor vessels are clearly visible. (c) Fluorescence video microscopy of peripheral and central tumor vascular beds. Arrowheads indicate hematopoietic stem cells (200X magnification). (d) Average number of hematopoietic stem cells per 200X microscopic field from (c) +/- SEM. (e) Cryosection of tumor immunostained with anti-mouse CD31 (green) at 200X and 400X magnification. Hematopoietic stem cells (arrowheads) are in or near blood vessels (arrows). Asterisk indicates P <0.05. Bar = 50 μm. Integrin α4β1 on human CD34 + stem cells is shown. (a) FACs profile for CD34, CD133 and integrin α4β1 on stem cells. (b) Adhesion of stem cells to CS-1 fibronectin +/− SEM in the presence of medium, anti-α4β1 (HP2 / 1) or control antibody (P1F6). (c) FACS profile for VCAM on EC (black) and non-specific IgG control (grey). (d) Adherence of stem cells to HUVEC in the presence of medium, anti-α4β1 (HP2 / 1) or control antibody (P1F6) +/− SEM. (e) Left, bright field / red fluorescence image of stem cells on EC. Right, red fluorescence image of stem cells on EC in the presence of anti-α4β1 (HP2 / 1) or control antibody (cIgG, P1F6). Asterisk indicates P <0.05. Integrin α4β1 and ligand in hematopoietic stem cell homing are shown. (a) Cryosections of mouse breast cancer or normal tissue (colon, left; heart, right) immunostained for CD31 (red) and VCAM or fibronectin (green). Arrowheads point to blood vessels. Yellow represents EC expression of VCAM / fibronectin. (b) Breast cancer (N202) from mice injected with hematopoietic stem cells (red, arrowheads) in the presence of anti-human α4β1 antibody or negative antibody (Cntrl) immunostained with anti-mouse CD31 (green, arrow) or Cryosection of Lewis lung cancer (LLC). (c-d) (c) Average number of hematopoietic stem cells per 200X microscopic field for N202 and (d) LLC tumors +/- SEM. Asterisk indicates P <0.05. Bar = 50 μm. Integrin α4β1 shows that hematopoietic stem cells promote homing from bone marrow. (a) Cryosections of LLC tumors from mice injected with EGFP + Lin− cells (green, arrowhead) and control antibody (cIgG) or anti-α4β1, immunostained with anti-CD31 (red, arrow). EGFP + blood vessels are yellow. (b) Average number of EGFP cells per 200X microscope field +/− SEM. (c) Cryosections of bFGF or VEGF saturated Matrigel from mice transplanted with Tie2LacZ bone marrow and treated with anti-α4β1 or control antibody (cIgG) stained to detect (200X) β-galactosidase. (d) Average number of LacZ + cells per 200X field from (c) +/- SEM: VEGF (black bar), FGF (white bar). (e) Cryosection from (d) immunostained for β-galactosidase (green) and CD31 (red). LacZ + / CD31 + blood vessels are yellow (arrows). (f) Average number of LacZ + / CD31 + vessels per 200X field +/− SEM. Asterisk indicates P <0.05. Bar = 50 μm.

Claims (31)

A method for altering the level of adhesion of hematopoietic progenitor cells to a target tissue comprising the following steps:
a) Supplying the following:
i) a population of cells comprising hematopoietic progenitor cells expressing integrin α4β1,
ii) a target tissue that is not a bone marrow endothelial tissue, and
iii) one or more agents that alter the specific binding of integrin α4β1 to integrin α4β1 ligand, and
b) treating one or more of the cell population and the target tissue with the agent under conditions for specific binding of the integrin α4β1 to the integrin α4β1 ligand, thereby Changing the level of adhesion to the target tissue.   2. The method of claim 1, wherein the treating step further comprises altering the level of transendothelial migration of hematopoietic progenitor cells.   2. The method of claim 1, wherein the treating step further comprises changing the level of differentiation of hematopoietic progenitor cells into a secondary cell type.   4. The method of claim 3, wherein the secondary cell type is not a bone marrow endothelial cell.   5. The method of claim 4, wherein the secondary cell type comprises one or more of mesenchymal cells, epithelial cells, muscle cells, nerve cells, immune cells, melanocytes, myoepithelial cells and embryonic cells.   Target tissue is vascular endothelium, muscle, neuron, tumor, inflammatory, peripheral blood, umbilical cord blood, heart, eye, skin, synovial fluid, tumor, lung, breast, prostate, neck, pancreas, colon, ovary, stomach, One or more of the tissues of the esophagus, oral cavity, tongue, gingiva, skin, liver, bronchi, cartilage, testicles, kidney, endometrium, uterus, bladder, spleen, thymus, thyroid, brain, neurons, gallbladder, eyes and joints The method of claim 1 comprising:   2. The method of claim 1, wherein the tissue is damaged.   2. The method of claim 1, wherein the tissue is ischemic.   2. The method of claim 1, wherein the target tissue comprises fibronectin.   2. The method of claim 1, wherein the target tissue comprises vascular tissue.   2. The method of claim 1, wherein the processing step is in vitro.   2. The method of claim 1, wherein the treatment step is in a mammalian subject in vivo.   13. The mammalian subject is selected from one or more of a subject having a disease, susceptible to disease, suspected of having the disease, and suspected of having the disease. the method of.   14. The method of claim 13, wherein the mammalian subject is a human.   14. The method of claim 13, wherein the disease is angiogenic.   14. The method of claim 13, wherein the disease is not angiogenic.   2. The method of claim 1, wherein the agent comprises an antibody.   18. The method of claim 17, wherein the antibody comprises an anti-integrin α4β1 antibody.   18. The method of claim 17, wherein the antibody comprises an anti-vascular cell adhesion molecule antibody.   18. The method of claim 17, wherein the antibody comprises an anti-fibronectin antibody.   2. The method of claim 1, wherein the ligand comprises a vascular cell adhesion molecule (VCAM).   2. The method of claim 1, wherein the ligand comprises fibronectin. A method for screening a test compound for altering the level of hematopoietic progenitor cell adhesion to a target tissue that is not bone marrow endothelial tissue, comprising the following steps:
a) Supplying the following:
i) a first composition comprising integrin α4β1,
ii) a second composition comprising one or more integrin α4β1 ligands, and
iii) test compound,
b) contacting the test compound with one or more of the first composition and the second composition under conditions for specific binding of the integrin α4β1 to the integrin α4β1 ligand; and
c) detecting a change in the level of specific binding of the integrin α4β1 to the integrin α4β1 ligand in the presence of the test compound as compared to in the absence of the test compound, thereby Identifying the level of adhesion of hematopoietic progenitor cells to the target tissue.   24. The method of claim 23, further comprising identifying the test compound as altering the level of transendothelial migration of hematopoietic progenitor cells.   24. The method of claim 23, further comprising identifying the test compound as altering the level of differentiation of hematopoietic progenitor cells into a secondary cell type.   26. The method of claim 25, wherein the secondary cell type is not a bone marrow endothelial cell.   27. The method of claim 26, wherein the secondary cell type comprises one or more of mesenchymal cells, epithelial cells, muscle cells, neural cells, immune cells, melanocytes, myoepithelial cells and embryonic cells.   Target tissue is vascular endothelium, muscle, neuron, tumor, inflammatory, peripheral blood, umbilical cord blood, heart, eye, skin, synovial fluid, tumor, lung, breast, prostate, neck, pancreas, colon, ovary, stomach, One or more of the tissues of the esophagus, oral cavity, tongue, gingiva, skin, liver, bronchi, cartilage, testis, kidney, endometrium, uterus, bladder, spleen, thymus, thyroid, brain, neurons, gallbladder, eye and joint 24. The method of claim 23, comprising:   24. The method of claim 23, wherein the contacting step is in vitro.   24. The method of claim 23, wherein the contacting step is in vivo in a non-human mammal. A method for isolating hematopoietic progenitor cells from tissue comprising the following steps:
a) Supplying the following:
i) a tissue containing hematopoietic progenitor cells, and
ii) an antibody that specifically binds to integrin α4β1;
b) treating the tissue with the antibody under conditions such that the antibody specifically binds to the integrin α4β1, and
c) isolating the integrin α4β1 that binds to the antibody, thereby isolating the hematopoietic progenitor cells.

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